Germline gain-of-function MMP11 variant results in an aggressive form of colorectal cancer

Matrix metalloproteinase-11 (MMP11) is an enzyme with proteolytic activity against matrix and nonmatrix proteins. Although most MMPs are secreted as inactive proenzymes and are later activated extracellularly, MMP11 is activated intracellularly by furin within the constitutive secretory pathway. It is a key factor in physiological tissue remodeling and its alteration may play an important role in the progression of epithelial malignancies and other diseases. TCGA colon and colorectal adenocarcinoma data showed that upregulation of MMP11 expression correlates with tumorigenesis and malignancy. Here, we provide evidence that a germline variant in the MMP11 gene (NM_005940: c.232C>T; p.(Pro78Ser)), identified by whole exome sequencing, can increase the tumorigenic properties of colorectal cancer (CRC) cells. P78S is located in the prodomain region, which is responsible for blocking MMP11's protease activity. This variant was detected in the proband and all the cancer-affected family members analyzed, while it was not detected in healthy relatives. In silico analyses predict that P78S could have an impact on the activation of the enzyme. Furthermore, our in vitro analyses show that the expression of P78S in HCT116 cells increases tumor cell invasion and proliferation. In summary, our results show that this variant could modify the structure of the MMP11 prodomain, producing a premature or uncontrolled activation of the enzyme that may contribute to an early CRC onset in these patients. The study of this gene in other CRC cases will provide further information about its role in CRC development, which might improve patient treatment in the future.     

Analysis of Intestinal Perfusion Data for Highly Permeable Drugs Using a Numerical Aqueous Resistance—Nonlinear Regression Method

Purpose. To develop, validate and apply a method for analyzing the intestinal perfusion data of highly permeable compounds using the Numerical Aqueous Resistance (NAR) theory and nonlinear regression (NAR-NLR) and to compare the results with the well-established Modified Boundary Layer (MBL) Analysis. Methods. The NAR-NLR method was validated and the results were compared to the MBL analysis results using previously reported cephradine jejunal perfusion data. Using the Single Pass Intestinal Perfusion (SPIP) method, the concentration dependence of intestinal permeability was investigated for formycin B, proline, and thymidine, three compounds reported to be absorbed by carrier-mediated transport processes. The MBL and NAR-NLR analyses were then applied to the three sets of SPIP data. Results. The results demonstrate that the intrinsic MBL transport parameters were highly variable and, in one case, the analyses failed to give a statistically significant Michaelis constant. The MBL mean dimensionless wall permeabilities (P*_w) were greater than the NAR-NLR P*_w and were also highly variable. In all cases, the NAR-NLR variability was significantly lower than the MBL variability. The extreme variability in the MBL-calculated P*_w is due to the sensitivity of P*_w when the fraction of unabsorbed drug (C_m/C_o) is low or, alternatively, when P*_w approached the aqueous permeability, P*_aq. Conclusions. The NAR-NLR method facilitates the analysis of intestinal perfusion data for highly permeable compounds such as those absorbed by carrier-mediated processes at concentrations below their K_m. The method also allows for the use of a wider range of flow conditions than the MBL analysis resulting in more reliable and less variable estimates of intestinal transport parameters as well as intestinal wall permeabilities.     

Structural diversity among anti-p-azophenylarsonate monoclonal antibodies from A/J mice: Comparison of Id and Id sequences

Amino acid sequence analyses were carried out on monoclonal anti-p-azophenylarsonate antibodies isolated from the ascites of mice carrying cell lines obtained from the fusion of A/J splenic lymphocytes with the myeloma cell line Sp2/0–Ag14. The partial primary structures of both heavy and light chains from seven idiotype negative hybridoma proteins are compared to those of six idiotype positive molecules. Amino-terminal amino acid sequences (40–47 residues) of heavy chains from molecules bearing the major cross-reacting idiotype, Id^CR, demonstrated 95% homology to each other. Similarly, aminoterminal sequences of Id^CR+light chains were homologous to each other. However, sequence variations were evident in individual antibodies in both framework and complementarity-determining regions, suggesting that a large family of molecules accounts for the major cross-reacting idiotype, as previously reported (Marshak-Rothstein et al., 1980b).

Heavy and light chains from seven Id^CR-negative monoclonal antibodies were subjected to amino-terminal (37–48 residues) amino acid sequence analysis. Four heavy chains were blocked to Edman degradation, but could be sequenced after enzymatic removal of the amino-terminal pyrrolidone carboxylic acid residue. In comparison with Id^CR-positive heavy chains, the Id^CR-negative heavy chains demonstrate greater diversity in both framework and complementarity-determining regions, with several different subgroups represented in contrast to the results from pooled serum Id^CR-positive antibodies (Capra et al., 1975). One of the seven Id^CR-negative light chains was blocked. The sequences of the remaining Id^CR-negative light chains exhibited marked variations in both framework and complementarity-determining regions, with different chain lengths in the first complementarity-determining region in several light chains.

Comparisons between the amino-terminal sequences of Id^CR-positive and Id^CR-negative monoclonal antibodies suggest that specific sequences in the first complementarity-determining regions of both heavy and light chains are not sufficient to account for the major cross-reacting idiotype. The structural basis for Id^CR in A/J mice is likely to be in other segments of the variable regions.     

Dipeptidyl carboxypeptidase 1 (DCP1) and butyrylcholinesterase (BCHE) gene interactions with the apolipoprotein E epsilon4 allele as risk factors in Alzheimer's disease and in Parkinson's disease with coexisting Alzheimer pathology

Alzheimer's disease (AD) and Parkinson's disease (PD) are genetically heterogeneous. Dipeptidyl carboxypeptidase 1 (DCP1) and butyrylcholinesterase (BCHE) genes may modify the risk of these disorders. We investigated whether common polymorphisms present in these genes operate as risk factors for AD and PD in Finnish subjects, independently or in concert with the apolipoprotein E ε4 allele (APOE ε4). Eighty late onset sporadic AD patients, 53 PD patients (34 of whom had concomitant AD pathology), and 67 control subjects were genotyped for the insertion (I)/deletion (D) polymorphism of DCP1 and the K variant of BCHE. In logistic regression analysis, the DCP1 *I allele in combination with APOE ε4 significantly increased the risk of AD (OR 30.0, 95% CI 7.3-123.7), compared to subjects carrying neither of the alleles. Similar analysis showed that the risk of AD was significantly increased in subjects carrying both the BCHE wild type (*WT/*WT) genotype and ε4 (OR 9.9, 95% CI 2.9-33.8), compared to those without this BCHE genotype and ε4. Further, the risk of PD with AD pathology was significantly increased for carriers of DCP1 *I and ε4 (OR 8.0, 95% CI 2.1-31.1). We thus conclude that, in Finns, interaction between DCP1 *I and ε4 increases the risk of AD as well as of PD with coexisting Alzheimer pathology, which underlines the importance of the DCP1 I/D polymorphism in the development of Alzheimer neuropathology, whereas the wild type BCHE genotype in combination with ε4 had a combined effect with regard to the risk of AD.


Keywords: Alzheimer's disease; Parkinson's disease; dipeptidyl carboxypeptidase 1; butyrylcholinesterase     

Cytochemical manifestations of short- and long-term activation of the rat brain dopaminergic system

Laboratory of Cytochemistry, Brain Research Institute, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR O. S. Adrianov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 7, pp. 41–42, July, 1991.     

Serine-containing 10-membered cyclodepsipeptides

As a part of a Research program aimed at studying synthesis and conformation of small ring peptides, the cyclization of diastereoisomeric N-phenylacetyl-seryl-propyl-proline tripeptides has been examined. Two 10- membered peptide lactones, and , have been isolated by treating the corresponding linear p-nitrophenyl esters with DBU in dry benzene. In these two compounds the serine lactone fragment (a common structural feature of several bioactive cyclodepsipeptides) is inserted into a highly strained small ring system. The conformation in the crystal of 5a and 5b has been studied by X-ray analysis. Both the 10-membered rings of 5a and 5b adopt an overall cis-cis-trans conformation in which the lactone junction is trans. The deviations from planarity of the peptide units vary from Δω= 30° for the DSer—Pro bond in 5b to Ao= 5-6° for the DSer-PrO bond in 5a and PrO—DPrO bond in 5b . The skeletal atoms of 5b , containing the PrO-DPrO sequence, are related by a pseudo-symmetry mirror plane passing through the Pro carbonyl and the opposite DSer C^βH₂ group. In both the molecules the exocyclic amide bond adopts an extended conformation with respect to the DSer-Pro ring junction; this arrangement gives rise to a C₅-type ring structure which is well evidenced in the case of 5a .     

肝癌相关性甘氨酰脯氨酸二肽氨基肽酶检测法

目的：进一步探讨血清肝癌相关性GPDA同工酶分离检测条件,提高其对原发性肝癌的诊断价值。方法：对分离血清肝癌相关性GPDA同工酶电泳体系的凝胶浓度及梯度、缓冲液的pH及离子强度、电泳的环境温度、标本 用量以及呈色条件等进行了逐项摸索,建立了稳定且分离效果好的GPDA同工酶检测方法。结果：采用三层阶段梯度聚丙烯酰胺凝胶体系将血清GPDA分为肝癌相关性的快带（GPDA－F）和相关性的慢带（GPDA－S）,肝癌相关性GPDA的检出率明显提高。结论：肝癌相关性血清GPDA检测法稳定可靠、敏感,适合于临床推广应用。     

Development and in Vitro Evaluation of Systems to Protect Peptide Drugs from Aminopeptidase N

Purpose. Develop and evaluate systems to prevent aminopeptidase N caused enzymatic degradation of perorally administrated peptide drugs. Methods. Bacitracin was covalently bound to the unabsorbable carrier matrix poly(acrylic acid) (paa) in order to avoid any dilution effects of the inhibitor in the intestine as well as systemic toxic side effects. The inhibitory effect of this conjugate, of neutralized paa and N-acetylcysteine was evaluated using a brush border membrane model. Results. Whereas within 6 h of incubation 65.3 ± 3.7 mol/1 of the substrate (L-leucine p-nitroanilide) was hydrolyzed under our assay conditions, this metabolism was reduced to 44.5 ± 6.3 mol/1 and 49.0 ± 8.8 mol/1 (n = 3–5; ± S.D.) using 1.5% bacitracin-polymer conjugate and 0.5% N-acetylcysteine, respectively. The same amount of bacitracin as immobilized to the polymer exhibited a comparably weaker inhibitory effect. Neutralized paa did not inhibit membrane bound aminopeptidase N. Covering the membrane with a thin mucus layer led to a significantly lowered inhibitory effect of all tested agents. Conclusions. The immobilization of enzyme inhibitors to a carrier matrix and the use of N-acetylcysteine as a novel inhibitor are promising strategies in order to overcome the enzymatic barrier caused by membrane bound peptidases. However the use of effective mucolytic agents seems to be a prerequisite.     

Structural Specificity of Mucosal-Cell Transport and Metabolism of Peptide Drugs: Implication for Oral Peptide Drug Delivery

The brush border membrane of intestinal mucosal cells contains a peptide carrier system with rather broad substrate specificity and various endo- and exopeptidase activities. Small peptide (di-/ tripeptide)-type drugs with or without an N-terminal -amino group, including -lactam antibiotics and angiotensin-converting enzyme (ACE) inhibitors, are transported by the peptide transporter. Poly-peptide drugs are hydrolyzed by brush border membrane proteolytic enzymes to di-/tripeptides and amino acids. Therefore, while the intestinal brush border membrane has a carrier system facilitating the absorption of di-/tripeptide drugs, it is a major barrier limiting oral availability of polypeptide drugs. In this paper, the specificity of peptide transport and metabolism in the intestinal brush border membrane is reviewed.