CYP1A2*1F and GSTM1 alleles are associated with susceptibility to porphyria cutanea tarda

Porphyria cutanea tarda (PCT) is a cutaneous porphyria with sporadic (type 1) and familial (type 2) subtypes, both resulting from decreased hepatic uroporphyrinogen decarboxylase (UROD) activity. Environmental and genetic factors are involved in the development of PCT, and genetic variants in the cytochrome P450 (CYP ) genes, CYP1A1 and CYP1A2, have been implicated. We investigated the association between PCT and variants in CYP1A1, CYP1A2 and CYP2E1, and the glutathione-S-transferase (GST ) genes, GSTM1 and GSTT1. PCT diagnosis was based on urinary or plasma porphyrin profiles. Patients were classified as type 1 or 2 PCT based on UROD mutation analysis. The CYP1A2*1F promoter A allele frequency was significantly higher (P < 0.022) and the A/A genotype frequency marginally higher in PCT patients overall (P < 0.057), with the A/A genotype significantly more common in type 1 PCT (P < 0.043). The presence of the wild-type GSTM1 allele also was associated significantly with PCT (P < 0.019). Neither hemochromatosis (HFE) mutations, tobacco smoking, hepatitis C and HIV infection, ethanol consumption, nor estrogen use were associated with these allelic variants. Age at onset was significantly lower in type 2 PCT patients (P < 0.001), as observed previously. Thus, positive associations between PCT and the CYP1A2*1F promoter A allele and A/A genotype and the wild-type GSTM1 allele indicates that these functional hepatic biotransformation enzymes are risk factors for the development of this disease.     

NGAL、KIM-1和PCT在脓毒症AKI中的标志物作用

探讨NGAL与KIM-1联合检测和PCT在重症监护病房重症患者中急性肾损伤(AKI)发生中的作用。选取2018年1月至2019年6月我院101例重症患者,其中脓毒症AKI组61例,非AKI组40例,通过分析NGAL、KIM-1和PCT在2组患者中表达水平变化情况,结合与ACR指标对比分析,评价NGAL、KIM-1和PCT在脓毒症急性肾损伤早期诊断中的价值。结果显示,所有脓毒症AKI患者均检测出明显更高的尿NGAL生物标志物水平(67.32μg/g Cr)。尿KIM-1和尿NGAL水平升高与患者ACR升高均呈正相关(p<0.001),而在脓毒症AKI患者中PCT和ACR之间观察到显著的负相关(r_s=-0.102 5, p=0.307)。通过Kruskal-Wallis检验发现,NGAL和KIM-1值显示出与脓毒症严重程度具有显著统计学意义,且直接成比例的关系(p≤0.01)。进一步检查NGAL、KIM-1和PCT标志物与病情发展的相关性表明,PCT值似乎与临床结果没有很强的相关性。尿KIM-1联合NGAL在早期检测脓毒症AKI中具有较大的预测价值;PCT是有希望的脓毒症标志物之一,但不足以提供可靠诊断依据,在肾功能下降的患者中通过PCT进行脓毒症的临床诊断需要更加谨慎。     

Molecular cloning of a malyl coenzyme A lyase gene from Pseudomonas sp. strain AM1, a facultative methylotroph

A genomic library containing HindIII partial digests of Pseudomonas sp. strain AM1 DNA was constructed in the broad-host-range cosmid pVK100. PCT57, a Pseudomonas sp. strain AM1 methanol mutant deficient in malyl coenzyme A lyase activity, was complemented to a methanol-positive phenotype by mobilization of the pVK100 library into PCT57 recipients with the ColE1/RK2 mobilizing plasmid pRK2013. Six different complemented isolates all contained a recombinant plasmid carrying the same 19.6-kilobase-pair Pseudomonas sp. strain AM1 DNA insert. Subcloning and complementation analysis demonstrated that the gene deficient in PCT57 (mcl-1) was located in a 1.6-kilobase-pair region within a 7.4-kilobase-pair EcoRI-HindIII fragment.     

Association of single nucleotide polymorphisms in SOD2, XRCC1 and XRCC3 with susceptibility for the development of adverse effects resulting from radiotherapy for prostate cancer

The objective of this study was to determine whether an association exists between certain single nucleotide polymorphisms (SNPs), which have previously been linked with adverse normal tissue effects resulting from radiotherapy, and the development of radiation injury resulting from radiotherapy for prostate cancer. A total of 135 consecutive patients with clinically localized prostate cancer and a minimum of 1 year of follow-up who had been treated with radiation therapy, either brachytherapy alone or in combination with external-beam radiotherapy, with or without hormone therapy, were genotyped for SNPs in SOD2, XRCC1 and XRCC3. Three common late tissue toxicities were investigated: late rectal bleeding, urinary morbidity, and erectile dysfunction. Patients with the XRCC1 rs25489 G/A (Arg280His) genotype were more likely to develop erectile dysfunction after irradiation than patients who had the G/G genotype (67% compared to 24%; P=0.048). In addition, patients who had the SOD2 rs4880 T/C (Val16Ala) genotype exhibited a significant increase in grade 2 late rectal bleeding compared to patients who had either the C/C or T/T genotype for this SNP (8% compared to 0%; P=0.02). Finally, patients with the combination of the SOD2 rs4880 C/T genotype and XRCC3 rs861539 T/C (Thr241Met) genotype experienced a significant increase in grade 2 late rectal bleeding compared to patients without this particular genotypic arrangement (14% compared to 1%; P=0.002). These results suggest that SNPs in the SOD2, XRCC1 and XRCC3 genes are associated with the development of late radiation injury in patients treated with radiation therapy for prostate adenocarcinoma.     

胃肠道穿孔感染性休克患者肠道菌群及其与血清C反应蛋白和降钙素原的相关性

目的探究胃肠道穿孔感染性休克患者肠道菌群及其与血清C反应蛋白(CRP)、降钙素原(PCT)的相关性。方法选取2017年1月到2019年6月辽宁省人民医院收治的110例胃肠道穿孔致感染性休克患者,按照APACHEII评分分为A组(APACHEII评分<20分,n=67)和B组(APACHEII评分≥20分,n=43)。另选52例同期体检正常的健康者作为正常组。检测各组对象血中免疫细胞数、肠道微生物及血清CRP、PCT水平,并分析患者肠道菌群与血清CRP、PCT水平的相关性。结果各组对象血液中白细胞、中性粒细胞、CD4^+细胞、CD4^+/CD8^+水平均为B组>A组>正常组,差异均有统计学意义(均P<0.05)。各组对象肠道肠杆菌数量为B组>A组>正常组,双歧杆菌数量及B/E值均为B组A组>正常组,差异均具有统计学意义(均P<0.05)。Pearson相关性分析显示,双歧杆菌数量、B/E值与血清CRP、PCT分别呈负相关(r=-0.624,-0.746;-0.638,-0.757,均P<0.05)。结论胃肠道穿孔感染性休克患者存在以双歧杆菌减少和肠杆菌增加为主要特点的肠道菌群失衡,且双歧杆菌数量和B/E值与血清CRP、PCT水平具有一定相关性,推测肠道菌群可能参与了疾病的发生和发展过程。     

Expression of glucose transporter isoforms (GLUT1, GLUT2) and activities of hexokinase,pyruvate kinase,and malic enzyme in preneoplastic and neoplastic rat renal basophilic cell lesions

Sequential changes in the expression of two glucose transporter isoforms (GLUT1, GLUT2), and in the activities of hexokinase, pyruvate kinase and malic enzyme during the development of rat renal basophilic cell tumors were studied using histochemical techniques. Early basophilic cell tubules are similar to proximal convoluted tubules (PCT) in their overall histochemical pattern, particularly in the expression of glucose transporters, suggesting that basophilic cell tubules and tumors derived from them arise from PCT. In comparison with PCT, basophilic cell tubules show slightly increased activities of all the enzymes studied. In basophilic cell tumors, markedly elevated hexokinase and pyruvate kinase activities are accompanied by a considerable reduction in the expression of GLUT2. GLUT1 expression is not found in basophilic cell tubules or PCT. Small basophilic cell tumors also do not express GLUT1, but GLUT1 is regularly expressed in several cell layers surrounding necrotic areas within large basophilic cell tumors. Our results indicate that increased glycolytic activity and reduced GLUT2 expression take place during the development of renal basophilic cell tumors.     

Transmission ratio distortion at the growth hormone gene (GH1) in bovine preimplantation embryos: An in vitro culture‐induced phenomenon?

The growth hormone gene (GH1) and its polypeptide product (GH) have a crucial role in reproduction, embryogenesis and general development. A polymorphism present in the fifth exon of the bovine GH1 gene (GH1 p.Leu127Val) has been associated with GH release and milk production in cattle. The objective of the present study was to examine the genotype frequencies of the GH1 p.Leu127Val polymorphism in bovine blastocysts produced in vitro and in vivo to determine if allelic variation of the GH1 gene affects embryo development and survival. A heterozygous (p.Leu127/Val127) sire was used for in vitro fertilization of oocytes of unknown maternal genotype (n = 104) and known maternal genotype (n = 115). PCR amplification and genotyping of the GH1 gene from Day 8 blastocysts derived from these fertilized oocytes demonstrated that there was significant over-representation from the expected Mendelian ratio of GH1 p.Leu127/Leu127 homozygotes from oocytes of known maternal genotype (P = 0.006). Contrary to this, analysis of in vivo-produced bovine blastocysts of known parental GH1 genotype (n = 69) did not reveal an overrepresentation of GH1 p.Leu127/Leu127 homozygotes. These results suggest that developing in vitro-produced embryos are exposed to a selection process, probably due to a less favorable culture environment, that acts to increase the number of GH1 p.Leu127/Leu127 homozygotes, thereby giving rise to the observed transmission ratio distortion (TRD) of GH1 genotypes when compared to in vivo produced embryos.     

Molecular cloning and characterization of a chemotactic transducer gene in Pseudomonas aeruginosa.

A Pseudomonas aeruginosa mutant, defective in taxis toward L-serine but responsive to peptone, was selected by the swarm plate method after N-methyl-N'-nitrosoguanidine mutagenesis. The mutant, designated PCT1, was fully motile but failed to show chemotactic responses to glycine, L-serine, L-threonine, and L-valine. PCT1 also showed weaker responses to some other commonly occurring L-amino acids than did the wild-type strain PAO1. A chemotactic transducer gene, denoted pctA (Pseudomonas chemotactic transducer A), was cloned by phenotypic complementation of PCT1. Nucleotide sequence analysis showed that the pctA gene encodes a putative polypeptide of 629 amino acids with a calculated mass of 68,042. A hydropathy plot of the predicted polypeptide suggested that PctA may be an integral membrane protein with two potential membrane-spanning regions. The C-terminal domain of PctA showed high homology with the enteric methyl-accepting chemotaxis proteins (MCPs). The most significant amino acid sequence similarity was found in the region of MCPs referred to as the highly conserved domain. The pctA gene was inactivated by insertion of a kanamycin resistance gene cassette into the wild-type gene, resulting in the same observed deficiency in taxis toward L-amino acids as PCT1. In vivo methyl labeling experiments with L-[methyl-3H]methionine showed that this knockout mutant lacked an MCP with a molecular weight of approximately 68,000.     

Amino-terminal truncation of procalcitonin, a marker for systemic bacterial infections, by dipeptidyl peptidase IV (DP IV)

Increased concentrations of procalcitonin (PCT) are found in the plasma of patients with thermal injury and in patients with sepsis and severe infection, making this molecule important as a diagnostic and prognostic marker in these diseases. Interestingly, only the truncated form of PCT, PCT(3-116), is present in the plasma of these patients. The enzyme responsible for this truncation is unknown as yet. Here, using capillary zone electrophoresis, mass spectrometry and Edman sequence analysis, we demonstrate that dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) is capable of catalyzing the hydrolysis of PCT(1-116), releasing the N-terminal dipeptide Ala-Pro. We hypothesize that PCT(3-116) is the result of the hydrolysis of PCT(1-116) by soluble DP IV of the blood plasma or by DP IV expressed on the surface of cells.     

Procalcitonin Levels Predict Acute Kidney Injury and Prognosis in Acute Pancreatitis: A Prospective Study

Background

Acute kidney injury (AKI) has been proposed as a leading cause of mortality for acute pancreatitis (AP) patients admitted to the intensive care unit (ICU). This study investigated the predictive value of procalcitonin (PCT) for AKI development and relevant prognosis in patients with AP, and compared PCT’s predictive power with that of other inflammation-related variables.

Methods

Between January 2011 and March 2013, we enrolled 305 cases with acute pancreatitis admitted to ICU. Serum levels of PCT, serum amyloid A (SAA), interleukin-6 (IL-6), and C reactive protein (CRP) were determined on admission. Serum PCT was tested in patients who developed AKI on the day of AKI occurrence and on either day 28 after occurrence (for survivors) or on the day of death (for those who died within 28 days).

Results

Serum PCT levels were 100-fold higher in the AKI group than in the non-AKI group on the day of ICU admission (p<0.05). The area under the receiver-operating characteristic (ROC) curve of PCT for predicting AKI was 0.986, which was superior to SAA, CRP, and IL-6 (p<0.05). ROC analysis revealed all variables tested had lower predictive performance for AKI prognosis. The average serum PCT level on day 28 (2.67 (0.89, 7.99) ng/ml) was significantly (p<0.0001) lower than on the day of AKI occurrence (43.71 (19.24,65.69) ng/ml) in survivors, but the serum PCT level on death (63.73 (34.22,94.30) ng/ml) was higher than on the day of AKI occurrence (37.55 (18.70,74.12) ng/ml) in non-survivors, although there was no significant difference between the two days in the latter group (p = 0.1365).

Conclusion

Serum PCT is superior to CRP, IL-6, and SAA for predicting the development of AKI in patients with AP, and also can be used for dynamic evaluation of AKI prognosis.     

Maternal cigarette smoking, metabolic enzyme polymorphism, and developmental events in the early stages of extrauterine life

The recent observation that maternal ACP1 genotype has an interactive effect with smoking on intrauterine development prompted us to search for a possible interaction effect between smoking and ACP1 genotype on haptoglobin (Hp) development in the neonatal period. ACP1 is a highly polymorphic protein tyrosine phosphatase involved in signal transduction of several growth factor receptors. The enzyme is composed of two isoforms, F and S. We studied 299 infants born in the Department of Obstetrics of the University Hospital of Rome La Sapienza. We found that an interaction between ACP1 genotype and smoking has an effect on haptoglobin development: A significant delay of haptoglobin development in infants born to smoking mothers is observed only in infants with the ACP1 *B/*B genotype, which shows the highest concentration of the ACP1 F isoform. The results indicate that the ACP1 genotype modifies the deleterious effects of smoking on development not only during intrauterine life but also during the early stage of extrauterine life.     

Microbial metabolism of C1 and C2 compounds. The role of glyoxylate, glycollate and acetate in the growth of Pseudomonas AM1 on ethanol and on C1 compounds

Succinate (or a product of succinate metabolism) is a catabolite repressor of some enzymes of the serine pathway (hydroxypyruvate reductase, serine-glyoxylate aminotransferase and glycerate kinase) but not of methanol dehydrogenase nor methylamine dehydrogenase. A mutant (PCT64) of Pseudomonas AM1, which is unable to grow on C(1) compounds, lacks glycerate kinase, showing that this enzyme is essential for the operation of the serine pathway. Mutant PCT48, unable to convert acetate into glycollate, has lost the ability to grow both on C(1) compounds and on ethanol. The properties of a third mutant (PCT57) show that Pseudomonas AM1 contains enzymes catalysing the conversion of acetate into glyoxylate. Evidence is presented that hydroxypyruvate reductase is involved in the oxidation of glycollate to glyoxylate during growth on ethanol. A scheme is proposed for the conversion of ethanol and of C(1) compounds into glyoxylate in which acetate (or a derivative) and glycollate are intermediates.     

Procalcitonin Predicts Real-Time PCR Results in Blood Samples from Patients with Suspected Sepsis

Background

Early diagnosis and rapid bacterial identification are of primary importance for outcome of septic patients. SeptiFast® (SF) real-time PCR assay is of potential utility in the etiological diagnosis of sepsis, but it cannot replace blood culture (BC) for routine use in clinical laboratory. Procalcitonin (PCT) is a marker of sepsis and can predict bacteremia in septic patients. The aim of the present study was to investigate whether PCT serum levels could predict SF results, and could help screening febrile patients in which a SF assay can improve the etiological diagnosis of sepsis.

Methods

From 1009 febrile patients with suspected sepsis, 1009 samples for BC, SF real-time PCR, and PCT determination were obtained simultaneously, and results were compared and statistically analysed. Receiver operating characteristic (ROC) curves were generated to determine the area under the curve and to identify which cut-off of PCT value produced the best sensitivity to detect SF results.

Results

Mean PCT values of sera drawn simultaneously with samples SF positive (35.42±61.03 ng/ml) or BC positive (23.14±51.56 ng/ml) for a pathogen were statistically higher than those drawn simultaneously with SF negative (0.84±1.67 ng/ml) or BC negative (2.79±16.64 ng/ml) samples (p<0.0001). For SF, ROC analysis showed an area under the curve of 0.927 (95% confidence interval: 0.899–0.955, p<0.0001). The PCT cut-off value of 0.37 ng/ml showed a negative predictive value of 99%, reducing the number of SF assays of 53.9%, still identifying the 96.4% of the pathogens.

Conclusion

PCT can be used in febrile patients with suspected sepsis to predict SF positive or negative results. A cut-off value of 0.37 ng/ml can be considered for optimal sensitivity, so that, in the routine laboratory activity, SF assay should not be used for diagnosis of sepsis in an unselected patient population with a PCT value <0.37 ng/ml.     

Adaptive mutations producing efficient replication of genotype 1a hepatitis C virus RNA in normal Huh7 cells

Despite recent successes in generating subgenomic RNA replicons derived from genotype 1b strains of hepatitis C virus (HCV) that replicate efficiently in cultured cells, it has proven difficult to generate efficiently replicating RNAs from any other genotype of HCV. This includes genotype 1a, even though it is closely related to genotype 1b. We show here that an important restriction to replication of the genotype 1a H77c strain RNA in normal Huh7 cells resides within the amino-terminal 75 residues of the NS3 protease. We identified adaptive mutations located within this NS3 domain and within NS4A, in close proximity to the essential protease cofactor sequence, that act cooperative to substantially enhance the replication of this genotype 1a RNA in Huh7 cells. These and additional adaptive mutations, identified through a series of iterative transfections and the selection of G418-resistant cell clones, form two groups associating with distinct nonstructural protein domains: the NS3/4A protease and NS5A. A combination of mutations from both groups led to robust replication of otherwise unmodified H77c genomic RNA that was readily detectable by northern analysis within 4 days of transfection into Huh7 cells. We speculate that these adaptive mutations favorably influence assembly of the replicase complex with host cell-specific proteins, or alternatively promote interactions of NS3/4A and/or NS5A with cellular proteins involved in host cell antiviral defenses.     

Isolation and characterization of serum procalcitonin from patients with sepsis

Procalcitonin (PCT) is one of the precursors in the synthesis of calcitonin in thyroidal C-cells and other neuroendocrine cells. PCT and other calcitonin precursors are elevated in the serum of many conditions leading to systemic inflammatory response syndrome. The measurement of PCT in patients suffering from severe bacterial infections is a useful tool for the diagnosis of sepsis. Furthermore, therapeutic decisions are often based on the increase or decline of serum PCT levels. PCT was reported to have 116 amino acids. The aim of our study was the determination of the primary structure of serum PCT from septic patients. Sera containing high PCT-concentrations (>100 ng/ml) were collected from 22 patients with severe sepsis and were pooled for further purification (12.7 μg total concentration of PCT). Pooled PCT was purified on a CT 21-immunoaffinity column, further purified by reversed phase HPLC, and the resulting pure PCT was digested with endoproteinase Asp-N. N-terminal Edman sequencing showed that the first two amino acids (Ala-Pro) of the proposed pro-peptide were missing. Further analyses by MALDI-TOF mass spectroscopy resulted in a distinct mass signal of 12640 Da ± 0.1%, which is in concordance with the theoretical molecular weight of the N-terminal truncated form (12628 Da). As opposed to previous suggestions, we could not detect any chemical modifications of PCT. In summary, we could demonstrate that PCT in the serum of septic patients is a peptide of only 114 amino acids, instead of the predicted 116 amino acids, lacking the N-terminal dipeptide Ala-Pro. This information on the primary structure of PCT might help in further studies on the physiological role of PCT during sepsis.     

Porphyria cutanea tarda: the etiological importance of mutations in the HFE gene and viral infection is population-dependent.

A number of factors, including increased iron stores and alcohol consumption, are known to be associated with the development of porphyria cutanea tarda (PCT) in susceptible individuals. Recent reports have described a significant association between inheritance of the C282Y and H63D mutations in the HFE gene, associated with genetic hemochromatosis (GH) and PCT. A strong association between hepatitis C virus infection and PCT has also been demonstrated, while case reports record a link between human immunodeficiency virus (HIV) and PCT. We have investigated the frequency of these factors in a racially-mixed population of patients with PCT in Cape Town, South Africa. 57 patients with PCT drawn from three ethnic groups were screened for the presence of the C282Y and H63D mutations linked to GH, and the prevalences were compared with corresponding healthy control populations. The seroprevalence of markers for HCV, hepatitis B (HBV) and HIV infection were examined in 28 of these. In the control populations, we found that both the C282Y and H63D mutations are highly prevalent in South Africans of European origin. In a population of mixed or Asian origin, the C282Y mutation is very rare whereas the H63D mutation is common. Neither mutation was encountered in any African subject. Both mutations are associated with PCT, but the association is dependent on the ethnic origins of the population to which the patient belongs. In contrast to other studies, HCV infection is numerically unimportant in PCT in our patients. HIV infection is increasingly encountered in our patients with PCT, but the strength of the association cannot be determined in view of the high background prevalence of HIV infection in some sectors of the South African population. The contribution of specific risk factors may be heavily dependent on the population from which patients are drawn, and care should be taken in extrapolating from observations in one racial or geographic population to any other.     

粘蛋白MUC1 568A/G SNP与辽宁地区人群胃癌遗传易感性的关系

为了探讨粘蛋白(MUC1)基因568位点A/G单核苷酸多态性与胃癌遗传易感性的关系, 采用序列特异性引物-聚合酶链反应(Sequence specific primers PCR, PCR-SSPs)检测来自辽宁地区人群138例胃癌患者及与其配比的131例对照个体MUC1 568 位点A/G多态性, 以ELISA法检测血清H. pylori IgG抗体。结果显示:(1)对照人群MUC1基因568位点AA、AG、GG 3种基因型分布频率分别为73.3%、22.1%、4.6%; (2)胃癌组MUC1 AA基因型携带频率显著高于正常对照组(P=0.03), 携带MUC1 AA基因型个体胃癌的发病风险增高到1.92倍; (3)以MUC1 AG+GG基因型并血清幽门螺杆菌(H. pylori)IgG抗体阴性的个体为对照, AG+GG基因型并H. pylori IgG抗体阳性个体、AA基因型并H. pylori IgG抗体阴性个体、AA基因型并H. pylori IgG抗体阳性个体胃癌患病风险增高, 但3组各组间差异均无统计学意义(P>0.05)。说明MUC1基因568位点A/G多态与胃癌的遗传易感性相关; MUC1 A/G基因多态性和H. pylori感染在胃癌发生发展过程未见交互作用。     

Analysis of insertion-deletion polymorphism in serotonin transporter gene in men of different ethnic appurtenance with acute alcoholic psychosis

Analysis of insertion-deletion polymorphism of serotonin vector gene (SLC6A4) was carried out in Russian and Tatar men with acute alcoholic psychosis. Significant interpopulation differences in the distribution of SLC6A4 genotype and allele frequencies were revealed. A relationship of L/S gene with the disease was detected in Russians and Tatars, but the presence of heterozygotic genotype was associated with early onset of chronic alcoholization and development of acute alcoholic psychosis in Tatars and with later alcoholization and disease development in Russians. The share of S/S genotype was significantly decreased in Russian patients aged over 35 years, which suggests selection aimed at elimination of short allele homozygotes among patients with this disease and probably different genetic prerequisites for early and late development of the disease in Russians. In Tatars aged over 35 years acute alcoholic psychosis is associated with L/L genotype (RR-3).