Study of the polymorphism of ovine alpha s1- and alpha s2-caseins by capillary electrophoresis

Polymorphism of ovine alpha s-caseins was studied by capillary electrophoresis at pH 3.0 +/- 0.1. Individual caseins (CN) were selected according to their genetic variants, as determined by PAGE and isoelectric focusing. The ovine caseins, containing different genetic variants of alpha s1-CN and alpha s2-CN, were fractionated by cation-exchange FPLC. The alpha s1-CN variants A, B and C, and the fast moving alpha s2-CN variant were identified by a capillary electrophoresis method. The fast moving alpha s2-CN variant, so-called for its behaviour in PAGE, also had a faster electrophoretic mobility than the common alpha s2-CN when analysed by the present technique. The capillary electrophoresis method gave excellent, rapid, automated separation of alpha s1-CN and alpha s2-CN variants and was suitable for screening studies.     

Technical note: Detection of bovine kappa-casein variants A, B, C, and E by means of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP)

OBJECTIVE: The purpose of this study was to describe the CT imaging features of preaortic esophageal veins in 10 patients with liver cirrhosis. CONCLUSION: Preaortic esophageal veins deriving from the paraesophageal varices course anterior to the descending aorta and drain into the hemiazygos vein. Preaortic esophageal veins are anatomically identical to extrinsic esophageal veins. The diameter of the veins we studied ranged from 1 to 8 mm (mean, 3.1 mm). Two preaortic esophageal veins were noted in each of two patients.     

G-Protein-dependent facilitation of neuronal alpha1A, alpha1B, and alpha1E Ca channels

Modulation of neuronal voltage-gated Ca channels has important implications for synaptic function. To investigate the mechanisms of Ca channel modulation, we compared the G-protein-dependent facilitation of three neuronal Ca channels. alpha1A, alpha1B, or alpha1E subunits were transiently coexpressed with alpha2-deltab and beta3 subunits in HEK293 cells, and whole-cell currents were recorded. After intracellular dialysis with GTPgammaS, strongly depolarized conditioning pulses facilitated currents mediated by each Ca channel type. The magnitude of facilitation depended on current density, with low-density currents being most strongly facilitated and high-density currents often lacking facilitation. Facilitating depolarizations speeded channel activation approximately 1.7-fold for alpha1A and alpha1B and increased current amplitudes by the same proportion, demonstrating equivalent facilitation of G-protein-inhibited alpha1A and alpha1B channels. Inactivation typically obscured facilitation of alpha1E current amplitudes, but the activation kinetics of alpha1E currents showed consistent and pronounced G-protein-dependent facilitation. The onset and decay of facilitation had the same kinetics for alpha1A, alpha1B, and alpha1E, suggesting that Gbeta gamma dimers dissociate from and reassociate with these Ca channels at very similar rates. To investigate the structural basis for N-type Ca channel modulation, we expressed a mutant of alpha1B missing large segments of the II-III loop and C terminus. This deletion mutant exhibited undiminished G-protein-dependent facilitation, demonstrating that a Gbeta gamma interaction site recently identified within the C terminus of alpha1E is not required for modulation of alpha1B.     

Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein

The specificity of the extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein was studied. Hydrolysis was monitored over time by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and urea-PAGE. The major pH 4.6-soluble peptides were isolated by high-performance liquid chromatography and identified by N-terminal amino acid sequencing and mass spectrometry. The time course of peptide formation indicated that His-8-Gln-9, Ser-161-Gly-162, and either Gln-172-Tyr-173 or Phe-23-Phe-24 were the first, second, and third bonds cleaved, respectively. Other cleavage sites included Asn-19-Leu-20, Phe-32-Gly-33, Tyr-104-Lys-105, Leu-142-Ala-143, Phe-150-Arg-151, Gln-152-Phe-153, Leu-169-Gly-170, and Thr-171-Gln-172. The proteinase had a broad specificity for the amino acid residues at the P1 and P'1 positions but showed a preference for hydrophobic residues at the P2, P3, P4, P'2, P'3, and P'4 positions.     

Distribution of alpha 1A, alpha 1B and alpha 1E voltage-dependent calcium channel subunits in the human hippocampus and parahippocampal gyrus

The distribution of voltage-dependent calcium channel subunits in the central nervous system may provide information about the function of these channels. The present study examined the distribution of three alpha-1 subunits, alpha 1A, alpha 1B and alpha 1E, in the normal human hippocampal formation and parahippocampal gyrus using the techniques of in situ hybridization and immunocytochemistry. All three subunit mRNAs appeared to be similarly localized, with high levels of expression in the dentate granule and CA pyramidal layer. At the protein level, alpha 1A, alpha 1B and alpha 1E subunits were differentially localized. In general, alpha 1A-immunoreactivity was most intense in cell bodies and dendritic processes, including dentate granule cells, CA3 pyramidal cells and entorhinal cortex pre-alpha and pri-alpha cells. The alpha 1B antibody exhibited relatively weak staining of cell bodies but stronger staining of neuropil, especially in certain regions of high synaptic density such as the polymorphic layer of the dentate gyrus and the stratum lucidum and radiatum of the CA regions. The alpha 1E staining pattern shared features in common with both alpha 1A and alpha 1B, with strong immunoreactivity in dentate granule, CA3 pyramidal and entorhinal cortex pri-alpha cells, as well as staining of the CA3 stratum lucidum. These findings suggest regions in which particular subunits may be involved in synaptic communication. For example, comparison of alpha 1B and alpha 1E staining in the CA3 stratum lucidum with calbindin-immuno-reactivity suggested that these two calcium channels subunits may be localized presynaptically in mossy fibre terminals and therefore may be involved in neurotransmitter release from these terminals.     

Localization and expression of the alpha1A-1, alpha1B and alpha1D-adrenoceptors in hyperplastic and non-hyperplastic human prostate

PURPOSE: To determine the expression and localization of the alpha1A-1, alpha1B and alpha1D-adrenoceptor (AR) subtypes in hyperplastic and non-hyperplastic human prostate tissue. MATERIALS AND METHODS: The expression of the alpha1-AR subtypes was examined at the mRNA level by quantitative solution hybridization, and at the protein level by immunohistochemistry using subtype selective antibodies. RESULTS: While the overall level of alpha1-AR mRNA was not significantly different between hyperplastic and non-hyperplastic tissue, there were significant differences in the ratio of the alpha1-AR subtypes expressed in the two tissue types. The most significant finding from these studies was the reduced expression of the alpha1b-AR mRNA in both glandular and stromal hyperplasia. By immunohistochemistry, the alpha1A-1-AR was detected in the stroma and not in the glandular epithelium. The alpha1B-AR was localized predominantly in the epithelium and was weakly present in the stroma. Lower levels of the alpha1B-AR were detected in the hyperplastic prostatic epithelium. The alpha1D-AR was detected in areas of stroma and was abundantly present in blood vessels. CONCLUSIONS: The alpha1A-1-, alpha1B- and alpha1D-AR subtypes are differentially localized in human prostate, and the expression levels of all three subtypes are altered in BPH. Alterations in a1-AR subtype expression (particularly the alpha1B-AR) in BPH cannot be solely attributed to changes in tissue morphometry resulting from hyperplasia and may be of significance in the pathogenesis of BPH.     

Cross-linking CD21/CD35 or CD19 increases both B7-1 and B7-2 expression on murine splenic B cells

Activation of the complement cascade and ligation of complement C3 receptors on B cells represent an important bridge between innate and Ag-specific acquired immunity. We show here that cross-linking of mouse CD21 (complement receptor type 2, CR2, C3d receptor) and CD35 (complement receptor type 1, CR1, C3b/C4b receptor) or co-cross-linking of CD21/CD35 and surface IgM rapidly up-regulates both B7-1 and B7-2 expression on murine resting splenic B cells. CD21/CD35-mediated up-regulation of both B7-1 and B7-2 expression is observed within 14 h, while other stimuli up-regulate only B7-2 but not B7-1 at this early time point. Consistent with the increase in B7 levels, BALB/c B cells on which surface IgM and CD21/CD35 have been co-cross-linked stimulate C57BL/6 T cells more effectively than controls. This CD21/CD35-enhanced allogeneic MLR is blocked nearly completely by anti-B7-2 mAbs and partially by anti-B7-1 mAbs. In addition, cross-linking of CD19, which is physically associated with CD21/CD35, leads to increased B7-1 and B7-2 expression. These data suggest that CD21/CD35 ligation results in enhanced B cell Ag presentation using costimulatory mechanisms shared with other activators and thus works cooperatively in this process. Rapid up-regulation of B7-1 expression, a unique response to CD21/CD35 and CD19 cross-linking, may be a particularly important effect of C3-containing ligands. We propose that CD21/CD35- and CD19-mediated B7-1 and B7-2 up-regulation is an important mechanism by which complement activation links innate and acquired immunity.     

Effect of immunization of rams against bovine inhibin alpha 1-26 on semen characteristics, scrotal size, FSH, LH and testosterone concentrations

White matter changes in Alzheimer's disease patients were the subject of the study. One hundred and seventeen cases clinically diagnosed according to NINCDS-ADRDA criteria as probable Alzheimer's disease were assessed. In computer tomography two types of pathological changes were observed; the so called leucoaraiosis and the perivascular focal alterations. Leucoaraiosis appeared in 20% of cases and focal changes were visible only in 4 cases A significant correlation was found between the presence of leucoaraiosis and the age and the presence of coronary disease. Hypertension, diabetes and brain atrophy did not seem to influence the occurrence of white matter alterations of either type.     

Determinant analysis of IgE and IgG4 antibodies and T cells specific for bovine alpha(s)1-casein from the same patients allergic to cow's milk: existence of alpha(s)1-casein-specific B cells and T cells characteristic in cow's-milk allergy

In an effort to clarify the etiology of milk allergy from the standpoint of allergen-specific immune reactions, we investigated the determinants of IgE, IgG4, and T cells specific for bovine alpha(s)1-casein from the same individual patients by using its synthetic peptides and cyanogen bromide-digested fragments. Alpha(s)1-casein is a major allergen in cow's milk, and its unique conformation enabled us to investigate the determinants of antibodies without consideration about missing the reactivities because of conformational changes. Nine patients were selected as subjects from among 129 milk-sensitive infants screened by ELISA to assess the anti-alpha(s)1-casein IgE levels in their sera. By using ELISA for epitope mapping, a C-terminal region of alpha(s)1-casein was identified as a common binding site for IgE from all of these patients, whereas those for anti-alpha(s)1-casein IgG4 were located in multiple regions of alpha(s)1-casein. We determined the specificities of seven alpha(s)1-casein-specific T-cell lines established from peripheral blood mononuclear cells of two of the patients. These T cells have been shown to secrete IL-4. All of the T-cell lines had different specificities to alpha(s)1-casein. However, a common amino acid residue use was found among the determinants of various T-cell lines from each patient. The results suggest that patients allergic to cow's milk have characteristic B cells recognizing a limited region of alpha(s)1-casein and secreting alpha(s)1-casein-specific IgE. These B cells may interact particularly with T cells recognizing determinants with a common structure.     

Characterization of the PP2A alpha gene mutation in okadaic acid-resistant variants of CHO-K1 cells

Okadaic acid (OA)-resistant variants of Chinese hamster ovary cells, clones CHO/OAR6-6 and CHO/OAR2-3, were isolated from a CHO-K1 culture. These variant cells were 17- to 26-fold more resistant to OA than the parental cells. The phosphorylase phosphatase activity of the variant cell extracts was 2- to 4-fold more resistant to OA than that of the parental cells in the presence of inhibitor 2, a specific inhibitor of type 1 protein serine/threonine phosphatase (PP1). Nucleotide sequencing of PP2A alpha (an isotype of PP2A catalytic subunit) cDNA demonstrated that both variants have a T-->G transversion at the first base of codon 269 (805 nt), which results in substitution of glycine for cysteine. We expressed in COS-1 cells a mutant PP2A alpha tagged with the influenza hemagglutinin epitope. The recombinant mutant PP2A alpha protein immunoprecipitated with an anti-influenza hemagglutinin antibody was more resistant than the wild type to OA, their IC50 values being 0.65 nM and 0.15 nM, and their IC80 values being 4.0 nM and 0.45 nM, respectively. The cysteine at residue 269 present only in highly OA-sensitive protein serine/threonine phosphatase catalytic subunit isozymes, PP2A alpha, PP2A beta, and PPX, is suggested to be involved in the binding of OA. CHO/OAR6-6 and CHO/OAR2-3 cells also overexpressed the P-glycoprotein, and the efflux of OA was more rapid. It is suggested that the PP2A alpha mutation in cooperation with a high level of P-glycoprotein makes the CHO-K1 variants highly resistant to OA.     

Distribution of the alpha1 to alpha6 chains of type IV collagen in bovine follicles

During follicular development the proliferative and differentiated state of the epithelioid granulosa cells changes, and the movement of fluid across the follicular basal lamina enables the formation of an antrum. Type IV collagen is an important component of many basal laminae. Each molecule is composed of three alpha chains; however, six different type IV collagen chains have been identified. It is not known which of these chains are present in the follicular basal lamina and whether the type IV collagen composition of the basal lamina changes during follicular development. Therefore, we immunolocalized each of the six chains in bovine ovaries using antibodies directed to the nonconserved non-collagenous (NC) domains. Additionally, dissected follicles were digested with collagenase to release the NC domains, and the NC1 domains were then detected by standard Western immunoblot methods. The follicular basal lamina of almost all primordial and preantral follicles was positive for all type IV collagen alpha chains. Colocalization of type IV collagen and factor VIII-related antigen allowed for discrimination between the follicular and endothelial basal laminae. Type IV collagen alpha1, alpha2, alpha3, alpha4, and alpha5 chains were present within the follicular basal lamina of only a proportion of antral follicles (17 of 22, 20 of 21, 15 of 18, 14 of 28, and 12 of 23, respectively), and staining was less intense than in the preantral follicles. Staining for the alpha1 and alpha2 chains was diffusely distributed throughout the theca in regions not associated with recognized basal laminae. The specificity of this immunostaining for alpha1 and alpha2 chains of type IV collagen was confirmed by Western immunoblots. As well as being detected in the basal lamina of approximately half of the antral follicles examined, type IV collagen alpha4 also colocalized with 3beta-hydroxysteroid dehydrogenase-immunopositive cells in the theca interna. Type IV collagen alpha6 was detected in the basal lamina of only one of the 16 antral follicles examined. Thus, the follicular basal lamina changes in composition during follicular development, with immunostaining levels being reduced for all type IV collagen chains and immunoreactivity for type IV collagen alpha6 being lost as follicle size increases. Additionally, immunoreactivity for alpha1 and alpha2 appears in the extracellular matrix of the theca as it develops.     

A functional role for specific spliced variants of the alpha7beta1 integrin in acetylcholine receptor clustering

The clustering of acetylcholine receptors (AChR) on skeletal muscle fibers is an early event in the formation of neuromuscular junctions. Recent studies show that laminin as well as agrin can induce AChR clustering. Since the alpha7beta1 integrin is a major laminin receptor in skeletal muscle, we determined if this integrin participates in laminin and/or agrin-induced AChR clustering. The alternative cytoplasmic domain variants, alpha7A and alpha7B, and the extracellular spliced forms, alpha7X1 and alpha7X2, were studied for their ability to engage in AChR clustering. Immunofluorescence microscopy of C2C12 myofibers shows that the alpha7beta1 integrin colocalizes with laminin-induced AChR clusters and to a much lesser extent with agrin-induced AChR clusters. However, together laminin and agrin promote a synergistic response and all AChR colocalize with the integrin. Laminin also induces the physical association of the integrin and AChR. High concentrations of anti-alpha7 antibodies inhibit colocalization of the integrin with AChR clusters as well as the enhanced response promoted by both laminin and agrin. Engaging the integrin with low concentrations of anti-alpha7 antibody initiates cluster formation in the absence of agrin or laminin. Whereas both the alpha7A and alpha7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the alpha7X2 extracellular domain were active. These results demonstrate that the alpha7beta1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the alpha7 chain, and that laminin, agrin, and the alpha7beta1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.     

碳含量对WC-9Ni-1Cr细晶硬质合金组织结构及性能的影响

通过添加W粉或C粉调整WC原料粉末的总碳含量(质量分数)为6.04%~6.16%,采用低压烧结法制备WC-9Ni-1Cr细晶硬质合金。采用光学金相显微镜、X射线衍射、扫描电镜等,研究碳含量对WC-9Ni-1Cr细晶硬质合金组织结构及性能的影响。结果表明:在WC-Ni系合金中添加适量的Cr元素,得到无磁WC-Ni硬质合金,并且其无磁特性不随合金中碳含量的变化而发生转变。WC粉末的总碳含量为6.04%~6.16%时WC-9Ni-1Cr细晶硬质合金为二相区的正常组织,只存在WC相和Ni相,没有石墨夹杂或η相;而且在此二相区范围内WC的碳含量变化对WC-9Ni-1Cr细晶硬质合金的耐腐蚀性没有明显影响。随WC粉末的碳含量增加,合金硬度(HRA)与密度都逐渐降低,但降低幅度较小,而合金的抗弯强度逐渐提高。碳含量由6.04%增加至6.16%时,抗弯强度由2 250 MPa提高到2 850 MPa,提高26.6%。     

Identification of the amino terminus of neuronal Ca2+ channel alpha1 subunits alpha1B and alpha1E as an essential determinant of G-protein modulation

We have examined the basis for G-protein modulation of the neuronal voltage-dependent calcium channels (VDCCs) alpha1E and alpha1B. A novel PCR product of alpha1E was isolated from rat brain. This contained an extended 5' DNA sequence and was subcloned onto the previously cloned isoform rbEII, giving rise to alpha1Elong whose N terminus was extended by 50 amino acids. VDCC alpha1 subunit constructs were co-expressed with the accessory alpha2-delta and beta2a subunits in Xenopus oocytes and mammalian (COS-7) cells. The alpha1Elong showed biophysical properties similar to those of rbEII; however, when G-protein modulation of expressed alpha1 subunits was induced by activation of co-expressed dopamine (D2) receptors with quinpirole (100 nM) in oocytes, or by co-transfection of Gbeta1gamma2 subunits in COS-7 cells, alpha1Elong, unlike alpha1E(rbEII), was found to be G-protein-modulated, in terms of both a slowing of activation kinetics and a reduction in current amplitude. However, alpha1Elong showed less modulation than alpha1B, and substitution of the alpha1E1-50 with the corresponding region of alpha1B1-55 produced a chimera alpha1bEEEE, with G-protein modulation intermediate between alpha1Elong and alpha1B. Furthermore, deletion of the N-terminal 1-55 sequence from alpha1B produced alpha1BDeltaN1-55, which could not be modulated, thus identifying the N-terminal domain as essential for G-protein modulation. Taken together with previous studies, these results indicate that the intracellular N terminus of alpha1E1-50 and alpha1B1-55 is likely to contribute to a multicomponent site, together with the intracellular I-II loop and/or the C-terminal tail, which are involved in Gbetagamma binding and/or in subsequent modulation of channel gating.     

Ontogenic development of the adenylyl cyclase enzyme and the alpha s, alpha i1 and alpha i2 G-protein regulatory subunits from rat prostate

STUDY OBJECTIVE: To compare the prophylactic administration of ondansetron with droperidol or placebo to determine its effectiveness in reducing postoperative nausea and vomiting after middle ear procedures. DESIGN: Prospective, randomized, double-blind study. SETTING: Inpatient otolaryngology service at a university medical center. PATIENTS: 120 ASA physical status I and II patients presenting for elective middle ear surgical procedures. INTERVENTIONS: Patients were randomly assigned to receive either placebo (Group 1), ondansetron 4 mg intravenously (IV) (Group 2), or droperidol 25 mcg/kg i.v. (Group 3) 10 minutes before induction of general anesthesia using thiopental 5 mg/kg i.v. with fentanyl 2 mcg/kg i.v. and maintenance anesthesia with isoflurane 1% to 2% end-tidal in a 50% air/oxygen mixture. MEASUREMENTS AND MAIN RESULTS: Total surgical, anesthesia, extubation, and postanesthesia care unit (PACU) occupancy times were recorded along with anesthesia recovery scores. The incidence and severity of nausea, vomiting, and pain along with rescue antiemetic administration, also were recorded. Similar assessments were made over the next 24 hours. Intergroup demographic data were similar except that the male to female ratio was higher in the ondansetron group. Stewart scores, reflecting emergence from anesthesia, were higher with ondansetron compared with droperidol. The incidence of nausea was similar between the groups but the severity was less after ondansetron therapy. More patients vomited after placebo than when given either droperidol or ondansetron. No intergroup differences were noted in the use of rescue antiemetics. Twenty-four hours later, more patients who received the placebo drug had nausea or vomited compared with either ondansetron or droperidol. CONCLUSIONS: Ondansetron 4 mg i.v. is as effective as droperidol and better than placebo in preventing nausea and vomiting in patients undergoing middle ear surgery. No cost advantage as determined by lower use of rescue antiemetics or shorter PACU times was noted after the prophylactic administration of ondansetron.     

Distinct alpha 7A beta 1 and alpha 7B beta 1 integrin expression patterns during mouse development: alpha 7A is restricted to skeletal muscle but alpha 7B is expressed in striated muscle, vasculature, and nervous system

The laminin binding alpha 7 beta 1 integrin has been described as a major integrin in skeletal muscle. The RNA coding for the cytoplasmic domain of alpha 7 integrin undergoes alternative splicing to generate two major forms, denoted alpha 7A and alpha 7B. In the current paper, we have examined the developmental expression patterns of the alpha 7A and alpha 7B splice variants in the mouse. The alpha 7 integrin expression is compared to that of the nonintegrin laminin receptor dystroglycan and to that of laminin-alpha 1 and laminin-alpha 2 chains. Alpha 7A integrin was found by in situ hybridization to be specific to skeletal muscle. Antibodies specific for alpha 7B integrin and in situ hybridization revealed the presence of alpha 7 mRNA and alpha 7B protein in the E10 myotome and later in primary and secondary myotubes. In the heart, alpha 7B integrin was not detectable in the endocardium or myocardium during embryonic and fetal heart development. Northern blot analysis and immunohistochemistry revealed a postnatal induction of alpha 7B in the myocardium. In addition to striated muscle, alpha 7B integrin was localized to previously unreported nonmuscle locations such as a subset of vascular endothelia and restricted sites in the nervous system. Comparison of the alpha 7 integrin expression pattern with that of different laminin isoforms and dystroglycan revealed a coordinated temporal expression of dystroglycan, alpha 7 integrin, and laminin-alpha 2, but not laminin-alpha 1, in the forming skeletal muscle. We conclude that the alpha 7A and alpha 7B integrin variants are expressed in a developmentally regulated, tissue-specific pattern suggesting different functions for the two splice forms.     

Disulphide arrangement in bovine caseins: localization of intrachain disulphide bridges in monomers of kappa- and alpha s2-casein from bovine milk

Naturally occurring monomeric kappa-casein and alpha s2-casein in bovine milk were purified by ion-exchange chromatography in order to localize potential intrachain disulphide bridges. Enzymic cleavage of the proteins followed by mass spectrometry and amino acid sequence analysis of cystine-containing peptides revealed the presence of an intrachain disulphide bond in both proteins.