Physicochemical characterization of the fifth (C5), sixth (C6), seventh (C7), eighth (C8) and ninth (C9) component of guinea pig complement

A physicochemical characterization of the purified guinea pig complement components C5 to C9 is given. For this purpose the sedimentation rate, the diffusion coefficient, the molecular weight and the isoelectric point were determined and compared with the values already known for the guinea pig and human complement system. For the determination of the physicochemical parameters gel filtration on Sephadex G-200, ultracentrifugation applying a sucrose density gradient and thin-layer isoelectric focusing were used. By comparing the values of the human and guinea pig complement a remarkable similarity is shown.     

Dipeptidyl peptidase IV (DP IV), a functional marker of the T lymphocyte system

Dipeptidyl peptidase IV (DP IV; E.C. 3.4.14.5), a plasma membrane structure of human T lymphocytes has been shown to be an important enzyme in the process of activation and proliferation of lymphocytes. In presence of specific inhibitors and antibodies against DP IV different functions of lymphocytes in vitro were found to be impaired. This holds true for mitogen and alloantigen induced DNA synthesis, immunoglobulin production and secretion, and interleukin-2 as well as interferon-gamma production. Studies of mitogen-induced expression of different activation markers (HLA class II antigen, 4F2, Tac) suggested that one of the functions of DP IV lies in overriding the cell cycle restriction point at G1. These data, together with other features of the DP IV, support the notion that this enzyme plays a key role in the modulation of lymphokine action by X-Pro- or X-Ala-directed limited proteolysis. Moreover the high frequency of DP IV susceptible bonds in different growth factors (e.g. IL-1, IL-2) and other biologically active peptides leads to the speculation that this peptidase is of more general significance to the regulation of cell growth.     

Discovery of a new biomarker for the mucopolysaccharidoses (MPS), dipeptidyl peptidase IV (DPP-IV; CD26), by SELDI-TOF mass spectrometry

Surface enhanced laser desorption/ionisation time of flight (SELDI-TOF) mass spectrometry has been used to search for new protein biomarkers in the plasma of patients with mucopolysacharidoses (MPS). Differences in the levels of some plasma proteins, particularly the apolipoprotein ApoCI, were observed between MPS patients and normal controls, using the different chromatographic surfaces (ProteinChips^®). ApoCI was identified by both its mass and by immunological techniques. In plasma, it exists in two forms, ApoCI and a truncated form which lacks two N-terminal amino acids, ApoCI′. In controls, the ratio of ApoCI′:ApoCI observed using the cation-exchange surface (CM10) was approximately 1:2 whereas in most MPS patients it varied from 1:1 to 1:0.8. The ratio of ApoCI′:ApoCI in plasma is determined by the activity of dipeptidyl peptidase IV, DPP-IV (also known as the leucocyte antigen CD26), which was found to be elevated up to 3-fold in MPS patients. The DPP-IV activity decreased in MPS I patients undergoing enzyme replacement therapy, indicating that it could be a useful biomarker for monitoring the efficacy of treatment in MPS disease. As DPP-IV has an important regulatory role in metabolism, it is possible that its elevation could cause some of the secondary pathology in MPS, and inhibition of DPP-IV might have a role in MPS therapy.     

A new supertypic HLA class II determinant (PL2) differentially expressed with DR7, DRw11(5), DRw13(w6), DRw14(w6), DR3, and DR2 and biochemically localized to DR7 molecules

A monoclonal antibody, PL2, has been produced that reacts with a new supertypic determinant expressed on the peripheral blood B lymphocytes and B-leukemic cells (B-CLL) from all individuals who are HLA-DR7 and some individuals who are HLA-DR5 positive. The genetic linkage of the PL2 determinant to the HLA region was demonstrated by family segregation studies. When cultured Epstein-Barr virus (EBV) transformed B cell lines were examined, PL2 was again found to be expressed on all cell lines homozygous for HLA-DR7 and the DRw11(5) subtype of HLA-DR5 positive cells, while one DRw12(5) cell line was negative, suggesting PL2 may distinguish between these DR5 subtypes. In addition, using the panel of EBV-transformed B-cell lines, PL2 was also found to be weakly expressed on HLA-DRw14(w6), -DRw13(w6), -DR3, and -DR2 positive cells but was completely absent from HLA-DR1 and -DR4 positive cells, and is probably absent also from DRw8- and DRw10-positive cells. From titration analysis and quantitative absorption studies the PL2 determinant was found to be expressed at quantitatively different levels in the following order: DR7 greater than DRw11, DRw14 greater than DRw13 greater than DR3 greater than DR2. The molecules carrying the PL2 determinant on DR7 cells have been characterized biochemically to be a subpopulation of HLA class II molecules recognized by the DR specific monoclonal antibody, L243. Furthermore, by two-dimensional gel analysis, PL2 immunoprecipitated only two of three beta chains associated with the DR-apha chain, which are the same two chains that carry the DR7 allodeterminants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence of the DNA-binding protein of a human subgroup E adenovirus (type 4): comparisons with subgroup A (type 12), subgroup B (type 7), and subgroup C (type 5)

The nucleotide sequence of the gene for the single-stranded DNA-binding protein of adenovirus type 4 (Ad4) has been determined. The gene codes for a protein of 512 amino acids. Comparison of the amino acid sequence with those previously determined for Ad5, Ad12, and Ad7 allowed identification of regions that are conserved between the four serotypes. These include stretches of 9, 9, and 12 amino acids in the carboxy-terminal domain of the protein; these sequences are similar to those identified in the single-stranded DNA-binding proteins of procaryotes as being important for interaction of the protein with single-stranded DNA. A conserved region of four amino acids in the amino-terminal domain is identical in sequence to a region of the SV40 large T antigen that has recently been implicated in the nuclear localization of the protein. Other conserved amino acids that may be important for the three-dimensional structure of the protein have also been identified. The overall homology between the DBPs of the four serotypes is 17.2% in the amino-terminal domain, 47.8% in the carboxy-terminal domain. Two-way comparisons between the DBPs of the four serotypes indicates that the DBP of Ad4 is most closely related to that of Ad7.     

Interactions of the streptococcal C5a peptidase with human fibronectin

Group B Streptococci (GBS) is a leading cause of sepsis and meningitis in neonates and immunocompromised adults in western countries. GBS do not bind to fibronectin (Fn) in solution, but will bind to Fn adsorbed onto a solid surface. The reason for the specificity of this binding is unknown. Single molecule force spectroscopy was used to test the hypothesis that GBS, through streptococcal C5a peptidase (ScpB) molecules present on the surface of the bacteria, binds to a motif created by the juxtaposition of multiple adjacent Fn molecules. Atomic force microscopy (AFM) topographical images of adsorbed Fn deposited from various Fn coating concentrations were used to determine the Fn surface concentration. ScpB was tethered to an AFM tip with all surface modifications characterized by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. At the lowest Fn coverages the probability of observing a ScpB–Fn binding event increased linearly with Fn surface coverage. As an Fn monolayer was reached the probability of a ScpB–Fn binding event occurring increased markedly (50 fold), with a concomitant increase in the rupture force from 17 pN to 33 pN. These results are consistent with the hypothesis that ScpB binds to a motif created by the juxtaposition of multiple Fn molecules.     

FLT4 Receptor Tyrosine Kinase Gene Mapping to Chromosome Band 5q35 in Relation to the t(2;5), t(5;6), and t(3;5) Translocations

FLT4 is a recently cloned receptor tyrosine kinase cDNA, which is characterized by seven immunoglobulin-like loops in its extracellular domain. We have previously mapped the FLT4 gene to chromosome segment 5q33-qter using somatic cell hybrids. Here we have refined the localization to band 5q35 by fluorescence in situ hybridization and show that the gene is translocated to chromosomes 2 and 6 in the t(2;5)(p23;q35) and t(5;6)(q35;p21) translocations, respectively, of Ki-1-positive lymphomas, as well as to chromosome 3 in the t(3;5)(q25.1;q34) translocation, which is occasionally found in myelodysplastic syndromes and acute myeloid leukemia. No evidence was obtained for a rearrangement or deregulation of the translocated FLT4 gene. We further show that abundant FLT4 mRNA expression occurs only in erythroid and megakaryoblastoid cell lines among nine leukemia cell lines studied. © 1993 Wiley-Liss, Inc.     

Non-reciprocal translocation (5;15), isodicentric (15) and Prader-Willi syndrome

A non-reciprocal translocation (5;15) and an isodicentric (15) resulting in trisomy 15pter----15q1?3 and monosomy 5qter [46,XY,-5,-15,+der(5)t(5;15) (5pter----5q35::15q13----15qter),+idic(15) (pter----q1?3::q1?3----pter)] was found in a 28-year-old profoundly retarded male resident of a state institution. Early developmental history and childhood and adult physical findings resembled those of Prader-Willi syndrome (PWS) patients. The parents' unbanded chromosomes were normal. Blood groups of parents and propositus were uninformative with regard to identifying gene deletions or duplications.     

Evolution of localization of the reactions of adenosine triphosphatase (Mg++-ATP-ase), 5'nucleotidase (5'nt), alkaline phosphatase (AP), and acid phosphatase (AcP) in developing rat testis. I. Physiological conditions

The experiments were performed upon the rats aged 1, 4, 7, 15, 30, 45, 60, 90 d, and 1,5 a. The behavior of the following reactions was described: for adenosine triphosphatase stimulated by Mg++(Mg++-ATP-ase), for 5'nucleotidase (5'Nt), for alkaline phosphatase (AP), for acid phosphatase (AcP). The first 3 are markers of the transport enzymes in cells, and the 4th is a marker of lytic processes. It was estimated on the basis of the examined reactions that a full metabolic maturity of the gonad was revealed since the 45th d of post-fetal life.     

Evolution of localization of reactions of adenosine triphosphatase (Mg++-ATP-ase), 5'nucleotidase (5'nt), alkaline phosphatase (AP), and acid phosphatase (AcP) in developing rat testis. II. After CdCl2 treatment

The described experiments show the influence of a single dose of cadmium chloride (1.5 mg CdCl2/kg body weight applied intraperitoneally) on development of the male gonads from the 1st d of post-fetal life to 1.5 a of life. In the case of the enzymes triggering transportation processes, adenosine triphosphatase stimulated by Mg++ (Mg++-ATP-ase), alkaline phosphatase (AP), and 5'nucleotidase (5'Nt), a considerable damages begin to appear in the 15th d of life whereas in the case of acid phosphatase (AcP) already in the 1st d of life whereas in the case of acid phosphatase (AcP) already in the 1st d of life. These damages increase with age reaching their maximum in the 45th d of life.     

Streptococcal C5a peptidase is a highly specific endopeptidase.

Compositional analysis of streptococcal C5a peptidase (SCPA) cleavage products from a synthetic peptide corresponding to the 20 C-terminal residues of C5a demonstrated that the target cleavage site is His-Lys rather than Lys-Asp, as previously suggested. A C5a peptide analog with Lys replaced by Gln was also subject to cleavage by SCPA. This confirmed that His-Lys rather than Lys-Asp is the scissile bond. Cleavage at histidine is unusual but is the same as that suggested for a peptidase produced by group B streptococci. Native C5 protein was also resistant to SCPA, suggesting that the His-Lys bond is inaccessible prior to proteolytic cleavage by C5 convertase. These experiments showed that the streptococcal C5a peptidase is highly specific for C5a and suggest that its function is not merely to process protein for metabolic consumption but to act primarily to eliminate this chemotactic signal from inflammatory foci.     

Enhanced Expression of CD80 (B7-1), CD86 (B7-2), and CD40 and Their Ligands CD28 and CD154 in Fulminant Hepatic Failure

To define a possible role for changes in the regulation of antigen presentation in fulminant hepatic failure (FHF), we studied the expression of co-stimulatory molecules CD80 (B7-1), CD86 (B7-2), and CD40 along with their ligands CD28 and CD154. We analyzed the liver tissue from patients with FHF (n = 18), chronic liver disease (n = 30), and acute hepatitis (n = 3) and from normal controls (n = 9) by immunohistochemistry and examined the temporal relationship between CD80/CD86 and CD40 expression and disease in the mouse models of galactosamine-lipopolysaccharide and galactosamine-tumor-necrosis-factor-induced FHF. In human controls, faint CD80/CD86 immunoreactivity was restricted to Kupffer cells, and CD40 expression was expressed on bile ducts, macrophages, and sinusoidal endothelial cells (SECs). In FHF, immunoreactivity for CD80 and CD86 was observed on significantly higher numbers of cells, including SECs. Increased CD80/CD86 expression corresponded to increased numbers of CD28-positive lymphocytes. The expression of CD40 was also clearly elevated on virtually all cell types in FHF. In both murine models, CD40 and CD80/CD86 expression was up-regulated before tissue damage could be detected. Our data suggest that up-regulated expression of co-stimulatory molecules might lead to an excessive antigen presentation in FHF as an early step in the pathogenesis before the onset of tissue damage.     

B族链球菌C5a肽酶蛋白表位的克隆、表达和免疫学分析

目的应用生物信息学方法预测B族链球菌C5a肽酶蛋白表位，结合基因工程手段进行表位重组、表达和免疫原性分析。方法用预测程序ProPred和ANTIGENIC预测B族链球菌C5a肽酶蛋白的表位，应用PCR技术扩增出编码该表位基因片段，克隆PCR产物构建重组质粒，测序验证。在大肠杆菌BL21（DE3）中诱导表达融合蛋白。表达的蛋白经质谱分析和Western blot鉴定。纯化该融合蛋白并免疫C57／BL小鼠，萃取GBS表面蛋白，双向琼脂扩散试验检测抗体水平。结果在SCPB中预测到1个既具有MHC结合肽特性又具有B细胞表位特征的肽段。重组和表达了这一肽段，质谱得出与SCPB蛋白的相似性分数为79，Western-blot证实能与抗SCPB的抗体反应，纯化后融合蛋白纯度〉90％。动物实验证实融合蛋白能产生特异性的抗GBS抗体。结论重组表位具有一定免疫原性。为相关蛋白的毒力机制研究和亚单位疫苗等方面的研究打下了良好的基础。     

Projections from the rat cuneiform nucleus to the A7, A6 (locus coeruleus), and A5 pontine noradrenergic cell groups

Stimulation of neurons in the cuneiform nucleus (CnF) produces antinociception and cardiovascular responses that could be mediated, in part, by noradrenergic neurons that innervate the spinal cord dorsal horn. The present study determined the projections of neurons in the CnF to the pontine noradrenergic neurons in the A5, A6 (locus coeruleus), and A7 cell groups that are known to project to the spinal cord. Injections of the anterograde tracer, biotinylated dextran amine in the CnF of Sasco Sprague-Dawley rats labeled axons located near noradrenergic neurons that were visualized by processing tissue sections for tyrosine hydroxylase-immunoreactivity. Anterogradely labeled axons were more dense on the side ipsilateral to the BDA deposit. Both A7 and A5 cell groups received dense projections from neurons in the CnF, whereas locus coeruleus received only a sparse projection. Highly varicose anterogradely labeled axons from the CnF were found in close apposition to dendrites and somata of tyrosine hydroxylase-immunoreactive neurons in pontine tegmentum. Although definitive evidence for direct pathways from CnF neurons to the pontine noradrenergic cell groups requires ultrastructural analysis, the results of the present studies provide presumptive evidence of direct projections from neurons in the CnF to the pontine noradrenergic neurons of the A7, locus coeruleus, and A5 cell groups. These results support the suggestion that the analgesia and cardiovascular responses produced by stimulation of neurons in the CnF may be mediated, in part, by pontine noradrenergic neurons.     

Differential expression of matrilysin-1 (MMP-7), 92 kD gelatinase (MMP-9), and metalloelastase (MMP-12) in oral verrucous and squamous cell cancer

Squamous cell carcinoma (SCC) of the oral cavity is a highly invasive tumour of stratified squamous epithelium that spreads through degradation of the basement membrane (BM) and extracellular matrix (ECM). There are currently no reliable tissue or serum markers to predict whether the tumour has metastasized at the time of diagnosis. Verrucous carcinoma (VC) of the oral cavity is a rare low-grade variant of oral SCC that penetrates into the subepithelial connective tissue. Many matrix metalloproteinases (MMPs), such as MMP-1, -2, -7, -9, -13, and -14, as well as integrin receptors have been implicated in cancer invasion. Integrin alphavbeta6 is induced in SCC and appears to be involved in up-regulation of MMP-9 expression by oral keratinocytes and promotion of their migration. The aim of this study was to investigate whether the pattern of MMP expression or that of alphavbeta6 integrin contributes to the differences in the biological behaviour of oral SCC and VC. The results show that the less aggressive nature of oral VC may be connected to its MMP expression profile. Typically, VCs were devoid of epithelial MMP-3, -7, -9, -12 and -13 expression, compared with SCCs. MMP-19 was expressed by epithelial keratinocytes in hyperproliferative areas of verrucous hyperplasia, VC, and SCC, but was absent in the invasive cancer cell nests of SCC. MMP-26 was expressed by hyperproliferative keratinocytes in VC as well as by invasive cancer cells in SCCs. MMP-10 was expressed widely in the epithelium of all SCC specimens. alphavbeta6 integrin expression was also detected in some cases of epithelial hyperplasia but was significantly more abundant in cancers at the invasive front. The absence of MMP-7, -9 and -12 from epithelial cells may serve as a good prognostic marker of non-invasive oral carcinoma. Blocking the activity of invasion-specific MMPs or alphavbeta6 integrin might offer novel therapeutic modalities in early-stage oral carcinoma.