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Blood choline has been proposed as a predictor of acute coronary syndrome (ACS), however different testing procedures might affect the choline concentration because the lysophospholipase D activity of autotaxin (ATX) can convert lysophosphatidylcholine to lysophosphatidic acid (LPA) and choline in human blood. Although the influences of ATX on LPA levels are well known in vivo and in vitro, those on choline have not been elucidated. Therefore, we established suitable sampling conditions and evaluated the usefulness of plasma choline concentrations as a biomarker for ACS. Serum LPA and choline concentrations dramatically increased after incubation depending on the presence of ATX, while their concentrations in plasma under several conditions were differently modulated. Plasma choline levels in genetically modified mice and healthy human subjects, however, were not influenced by the ATX level in vivo, while the plasma LPA concentrations were associated with ATX. With strict sample preparation, the plasma choline levels did not increase, but actually decreased in ACS patients. Our study revealed that ATX increased the choline concentrations after blood sampling but was not correlated with the choline concentrations in vivo; therefore, strict sample preparation will be necessary to investigate the possible use of choline as a biomarker.

ObjectivesThree multicentre studies of reference intervals were conducted recently in Japan. The Committee on Common Reference Intervals of the Japan Society of Clinical Chemistry sought to establish common reference intervals for 40 laboratory tests which were measured in common in the three studies and regarded as well harmonized in Japan.MethodsThe study protocols were comparable with recruitment mostly from hospital workers with body mass index ≤28 and no medications. Age and sex distributions were made equal to obtain a final data size of 6345 individuals. Between-subgroup differences were expressed as the SD ratio (between-subgroup SD divided by SD representing the reference interval). Between-study differences were all within acceptable levels, and thus the three datasets were merged.ResultsBy adopting SD ratio ≥0.50 as a guide, sex-specific reference intervals were necessary for 12 assays. Age-specific reference intervals for females partitioned at age 45 were required for five analytes. The reference intervals derived by the parametric method resulted in appreciable narrowing of the ranges by applying the latent abnormal values exclusion method in 10 items which were closely associated with prevalent disorders among healthy individuals. Sex- and age-related profiles of reference values, derived from individuals with no abnormal results in major tests, showed peculiar patterns specific to each analyte.ConclusionCommon reference intervals for nationwide use were developed for 40 major tests, based on three multicentre studies by advanced statistical methods. Sex- and age-related profiles of reference values are of great relevance not only for interpreting test results, but for applying clinical decision limits specified in various clinical guidelines.

Objectives Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid mediator. Although the plasma S1P concentration is reportedly determined by cellular components, including erythrocytes, platelets, and vascular endothelial cells, the possible involvement of other factors, such as serum sphingomyelin (SM) and autotaxin (ATX), remains to be elucidated. Design and methods We measured S1P using high-performance liquid chromatography (HPLC), SM and lysophosphatidic acid (LPA) using enzymatic assays, ATX antigen using a two-site enzyme immunoassay, and ATX activity using a lysophospholipase D activity assay. To fractionate the lipoproteins, plasma samples were separated using fast protein liquid chromatography (FPLC) utilizing a Superose 6 column. Results The plasma S1P level was positively correlated with the levels of SM and lysophosphatidylcholine, but not with the level of phosphatidylcholine. Although SM was present in the very low-density lipoprotein (VLDL) fraction, neither the plasma S1P level nor the SM level was affected by feeding. The plasma S1P level was negatively correlated with the ATX activity. Although the incubation of 100 μmol/L of sphingosylphosphorylcholine (SPC) with the serum resulted in a significant increase in the S1P level because of the presence of ATX, the physiological concentration of SPC did not mimic this effect. Conclusion The plasma S1P level was affected by the serum SM level, while the possibility of ATX involvement in the increase in the plasma S1P level was considered to be remote at least in healthy human subjects.

Clinical laboratory data used in medical examinations and health screening need to have a guaranteed analytical reliability. To ensure the reliability of measurement data, each constituent is required to be compatible with its traceability chain, and any constituent whose traceability chain has yet to be established is required to be appropriately harmonized in the current measurement system. The inter-laboratory reproducibility of standardized measurement values obtained from the external quality assessments conducted by the Japan Medical Association and Japanese Association of Medical Technologists was estimated to evaluate the analytical reliability of clinical tests in Japan. The estimated inter-laboratory reproducibility was then compared with the permissible error limits which have been reported domestically and internationally based on inter- and intra-individual biological variations of healthy subjects. The results showed that most of the measurement uncertainties were sufficiently lower than the permissible limits. This study proposes that the measurement uncertainty of the standardized measurement method has the potential to be a new assessment standard for analytical reliability.

Background Since sphingosine-1-phosphate (Sph-1-P) plays an important role as an extracellular mediator through interaction with specific cell surface receptors, especially in the area of vascular biology and immunology/haematology, determination of its plasma concentration may become important from the clinical viewpoint. Thus, we attempted to develop a method of measuring the plasma Sph-1-P concentration for use in the clinical laboratory setting. Methods After two-step lipid extraction, Sph-1-P was coupled with o-phthaldialdehyde, and the resultant fluorescent derivative was separated by high-performance liquid chromatography. C17-Sph-1-P was used as the internal standard, instead of dihydrosphingosine-1-phosphate, which had been used previously for the same purpose but was actually detected in plasma. Results Our procedures for preparing the plasma samples and assay Sph-1-P were found to be satisfactory for clinical laboratory testing. The plasma Sph-1-P concentrations were significantly higher in men (413.1 ± 52.0 nmol/L; mean ± SD) than in women (352.4 ± 39.7 nmol/L). Unexpectedly, strong positive correlations were found between the plasma Sph-1-P concentration and red blood cell (RBC)-related parameters, rather than platelet-related parameters. Conclusions Our present study confirmed the possibility of the clinical introduction of plasma Sph-1-P measurement, and in addition, suggested that RBCs may be involved in the regulation of plasma Sph-1-P concentrations.

Tokyo University of Technology established a Faculty of Health Sciences, consisting of Departments of Nursing, Clinical Engineering, Physical Therapy, and Occupational Therapy in 2000, and established the Department of Medical Technology in 2014. Now, more than half a century after medical technologists' education started in 1959, medical technologists' training schools exist in 80 facilities, with universities making up 52 schools. On this occasion, we consider the role of medical technologists' training in the future. The role of the clinical laboratory is to provide quick, timely, and useful medical information for the diagnosis, treatment, and prevention of diseases. Advances in medical technology in recent years have been marked. Today, the appropriateness of clinical laboratory services and systems is evaluated, focusing on the fulfillment of social and technical requirements, and this necessitates medical institutions to disclose their purposes and goal achievement levels. For the advancement of clinical laboratories, the process of assessing their achievements is important, which will maintain and improve reliability and promote development of the organization. Self-assessment is ongoing, and the improvement is required in clinical laboratories. Medical technologists in the future must be able to adapt flexibly to these situations. The nature of medical technologists' training is also similar, as they must acquire practical skills on-site, realize their self-growth potential, and understand that human power is important.

BACKGROUNDThe Japanese Committee for Clinical Laboratory Standards (JCCLS) has developed a multianalyte conventional reference material (MacRM) for nationwide standardization of laboratory measurements.METHODSTo prepare the MacRM, pooled sera were obtained from healthy Japanese individuals. Target values of the pooled sera for 30 analytes were assigned on the basis of the measurement results of 45 certified clinical laboratories whose calibration was verified by measuring certified reference materials (CRMs) provided by the National Institute of Standards and Technology, the Institute for Reference Materials and Measurements, and JCCLS. Commutability of MacRM was assessed by comparison with results for 150 individual inpatients at Fukuoka University Chikushi Hospital. Survey samples were prepared by essentially the same method for MacRM but without target values. The survey samples were used to assess agreement among 165 laboratories that used various assay kits and platforms calibrated with the MacRM.RESULTSThe commutability of MacRM was confirmed for 30 analytes with sera from 150 individual patients. The imprecision (CV) of measurements of survey samples (high and low concentrations) among the 165 laboratories was 0.4%–10.0%. Twenty-six of 30 analytes were within the goals for interinstitutional allowable bias. An aliquot of MacRM stored frozen at −80 °C remained stable for ≥4 years.CONCLUSIONSThe MacRM was successfully applied as a calibrator to achieve nationwide standardization for 30 analytes measured by 165 laboratories that used various methods from different manufacturers.

The LIM domain is a protein-protein interaction motif critically involved in a variety of fundamental biological processes, including cytoskeletal organization, cell lineage specification, and organ development. In this study we examined the expression of the LIM proteins paxillin and Hic-5 in adult human tissues by immunohistochemistry and immunoblotting. Paxillin expression was widespread and observed both in non-muscle and muscle tissues. Of the latter, paxillin was mainly expressed in multinuclear striated muscle. In contrast, Hic-5 showed restricted expression and was expressed in muscle tissues, mainly in mononuclear smooth muscle. Taken together with previous findings, it appears likely that the counterbalance between paxillin and Hic-5 may be deeply involved in muscle differentiation.

Hic-5 and paxillin, members of the LIM protein family, have been shown to be localized in focal adhesion and to have a role in integrin-mediated signalling. In the present study we examined the involvement of Hic-5 in human platelet activation: platelets express Hic-5 but not paxillin, whereas human umbilical-vein vascular endothelial cells and MEG-01 cells express mainly paxillin. When platelets were stimulated with thrombin, collagen or the stable thromboxane A(2) analogue U46619, Hic-5 was markedly tyrosine-phosphorylated, in a manner dependent on integrin alphaIIbbeta3-mediated aggregation. In addition, direct activation of protein kinase C with PMA resulted in tyrosine phosphorylation of Hic-5 only when platelets were fully aggregated with the exogenous addition of fibrinogen. Furthermore, PMA-induced Hic-5 tyrosine phosphorylation was also observed when platelets adhered to immobilized fibrinogen. In studies on immunoprecipitation and immunodepletion, Hic-5 seemed to associate with proline-rich tyrosine kinase 2 (Pyk2) but only marginally with focal adhesion kinase. When platelets were stimulated with thrombin, both Hic-5 and Pyk2 translocated to the cytoskeleton from the cytosol and membrane fractions in a manner dependent on alphaIIbbeta3-mediated aggregation. Finally, on stimulation with PMA, Hic-5, as well as Pyk2, translocated to the cell periphery, where a meshwork of actin filaments assembled after adhesion to immobilized fibrinogen. Our results suggest that Hic-5 might be important in platelet aggregation and adhesion, in a manner dependent on alphaIIbbeta3-mediated outside-in signalling, through association with Pyk2.

We report a case of Waldenstrom's macroglobulinemia with prominent bleeding tendency; laboratory investigation revealed an elevated activated partial thromboplastin time. Further laboratory evaluation showed circulating factor VIII anticoagulant, deemed polyclonal IgG, with a titer of 700 Bethesda Units/ml. The factor VIII inactivation kinetics of the patient plasma were identical to those of a type II inhibitor, and the inhibitor was found to recognize the A2 domain of the factor VIII heavy chain. Apparently, paraprotein is not always the cause of reduced activity of coagulation factors in neoplastic dysproteinemias.

電気泳動法による血清蛋白分画値に関する全国コントロール・サーベイを実施し, 施設内・施設間誤差変動の大きさを推定するとともに, 変動要因の解析を行った. その結果, 施設間総誤差変動はアルブミン分画で約3%, グロブリン分画で約11～14%の変動係数を示した. これら変動の内訳として, 施設内変動を上回る施設間変動を認め, また, 施設内においては日内変動に対して有意な日間変動を認めた. 分析上の要因としては, セ・ア膜の種類により, セパラックス膜は同SP膜に比べα1, α2-glo分画で高値を示す傾向にあり, 逆にγ-glo分画で2%ほど低値を示した. また, 自動分析装置の機種間においても測定値やその誤差変動の大きさが異なる傾向も認められた. 一方, このような誤差特性をもつ蛋白分画検査の施設内精度管理法として, x-Rs-R管理図法の適用を試み, その有用性を確認した.

Cholesteryl ester transfer protein (CETP) is one of the factors that regulate plasma levels of HDL-cholesterol. To identify the factors that may regulate CETP activity, and to determine to what extent CETP is correlated with physiologic concentrations of lipoprotein, we performed an epidemiologic study in 586 healthy volunteers (317 males and 269 females mean age 52.2 +/- 10.9 years). CETP activity in these subjects was 192.96 +/- 48.73 (mean +/- S.D.) nmol/ml/h and distributed to a wide range (60-450 nmol/ml/h). Using multiple regression analysis, we found significant positive correlations between CETP activity and LDL-cholesterol (P < 0.03), apolipoprotein (apo) E (P < 0.005) and LCAT activity (P < 0.001). CETP activities showed significant negative correlation with apo A-I (P < 0.03). However, CETP activity showed no significant correlation either with HDL cholesterol or with apo B. One-way layout analysis of variance showed that alcohol drinking and cigarette smoking significantly reduced CETP activity, but there was no significant association between CETP activity and body mass index. Although CETP activities were significantly higher in females than in males (P < 0.001), multiple regression analysis showed no correlation between CETP activity and age in either the males or the females. Our results suggest that CETP activity regulates the concentration of apo A-I and LDL-cholesterol, and that such activity may be influenced by gender, alcohol consumption and cigarette smoking.

http://dx.doi.org/10.1016/j.cca.2011.09.016, Background : International external quality assessments have shown variation in results of urinary albumin among various immunoassays. A well-defined candidate reference material for urine albumin (cRM-UA) was prepared to improve standardization. Methods : cRM-UA was prepared from a commercially available preparation of human serum albumin by using gel-filtration HPLC. The value was assigned by transfer from ERM-DA470 using immunoassay systems qualified based on the linearity and variability observed in dilution tests of pooled urine and the calibrators. Effectiveness of recalibration using the cRM-UA was evaluated by measuring 129 urine specimens. Results : The cRM-UA had a monomeric albumin peak which accounted for 98.9% of the total area by gel filtration HPLC. The lyophilized preparation of the cRM-UA had suitable homogeneity, and short- and long-term stability. Nine of 14 immunoassays met the criteria were used for value assignment. The assigned concentration was 225.1 ± 9.11 mg/l [mean ± U: expanded uncertainty with k = 2] when reconstituted with 3.00 ml of purified water on weight basis. Recalibration of 7 qualified immunoassays using the cRM-UA resulted in between-method CV of 6.6%. Conclusions : The cRM-UA was successful in achieving standardization of urine albumin results among 7 immunoassays which possess performance attributes representing uniform reactivity to both cRM-UA and clinical urine samples.

The International Organization for Standardization (ISO) developed a guide to the expression of uncertainty in measurement (GUM). The purpose of such guidance is to provide a basis for the international comparison of measurement results. In this study, we propose a basic protocol to evaluate and express uncertainty in measurement for routine test results in the clinical laboratory. We also sought to investigate the effects of measurement errors on the evaluation of biological variations in healthy subjects. To this end, we analyzed the allowable limits of analytical error which guarantee the reliability of reference intervals for the interpretation of clinical laboratory data. As a conclusion, we suggest that 1/2 or less of biological intraindividual variations is an appropriate criterion for an allowable limit of uncertainty to be applied in health check-ups, and this value is in agreement with previous reports. If this criterion as a marker for intra laboratory imprecision is met, it suggests that a given institute is able to evaluate time series changes in follow-up of individual data. If the reference interval of laboratory data for disease screening is shared by different institutes, it is suggested that a criterion of 1/4 or less of a biological inter- pulse intra-individual variation is appropriate. This criterion appears to be the goal for analytical inter-laboratory variations.

Background Lysophosphatidic acid (LPA) plays important roles in a variety of biological responses, especially in the area of vascular biology, and the determination of its plasma concentration is believed to be important. Several mechanisms are known to be involved in the metabolism of LPA. Methods To identify factors that may determine the plasma concentrations of this important bioactive lipid, we examined its concentrations using an enzymatic cycling assay and related parameters in 146 healthy subjects. Results The LPA concentration was significantly higher in women (mean ± SD, 0.103 ± 0.032 μmol/L; n = 47) than in men (0.077 ± 0.026 μmol/L; n = 99). A multiple regression analysis showed a strong positive correlation between the plasma LPA concentration and serum lysophospholipase D (lysoPLD) activity, while the LPA concentration was correlated with the plasma lysophosphatidylcholine (LPC) concentration only in men. Other lipid-related parameters were only slightly correlated or were not correlated with the LPA concentration. Conclusions Our findings suggested that conversion from LPC by lysoPLD might be the major route for LPA production in plasma.

Platelet-derived mediators may play an important role in the development of renal diseases through interaction with glomerular mesangial cells (MCs), and we, in this study, examined the effect of sphingosine 1-phosphate (Sph-1-P), a bioactive lipid released from activated platelets, on the contraction of MCs. Sph-1-P was found to induce MC contraction through mediation of Rho kinase both in cell shape change and collagen gel contraction assays. The specific antagonist of the Sph-1-P receptor S1P 2 inhibited the response. Similar results were obtained when the supernatant from activated platelet suspensions were used instead of Sph-1-P. Our findings suggest that platelet-derived Sph-1-P may be involved in MC contraction via S1P 2 and that regulation of this receptor might be useful therapeutically.

The genotype of hepatitis C virus (HCV) and the amount of HCV RNA are often used to predict the efficacy of interferon (IFN) therapy on chronic hepatitis C. In addition to these factors, there may be several factors related to the host. Therefore, the authors undertook a retrospective study in which physical findings and laboratory data before therapy were evaluated by multiple logistic analysis. Two-hundred and five cases with chronic hepatitis C treated with interferon were analyzed in this study. Sustained virological response was observed with 68 cases. Multiple logistic analysis was performed with 29 explanatory variables including HCV genotype, HCV RNA, IFN types, and total dose, along with gender, age, alcohol consumption, body mass index (BMI), histological findings of liver biopsy, platelet counts, and laboratory data of serum enzymes. Analysis on the factors that correlated well with therapeutic efficacy revealed that genotype 2a, 2b showed higher therapeutic responses than genotype 1b with reference to HCV genotypes, and higher IFN dose or lower HCV RNA levels gave better results. With reference to host factors, higher total protein level, lower levels of BMI, total bilirubin, and aspartate aminotransferase were highly correlated with therapeutic efficacy. HCV genotypes and HCV RNA levels have been already identified as prognostic factors. However, the high correlation values of BMI and the total protein level are new findings. It is suggested that probability estimation of therapeutic effects using the logistic regression equation may be a good tool for predicting therapeutic efficacy of IFN therapy on individual cases.

Many external quality assessment schemes (EQAS) are performed to support quality improvement of the services provided by participating laboratories for the benefits of patients. The EQAS organizer shall be responsible for ensuring that the method of evaluation is appropriate for maintenance of the credibility of the schemes. Procedures to evaluate each participating laboratory are gradually being standardized. In most cases of EQAS, the peer group mean is used as a target of accuracy, and the peer group standard deviation is used as a criterion for inter-laboratory variation. On the other hand, Fraser CG, et al. proposed desirable quality specifications for any imprecision and inaccuracies, which were derived from inter- and intra-biologic variations. We also proposed allowable limits of analytical error, being less than one-half of the average intra-individual variation for evaluation of imprecision, and less than one-quarter of the inter- plus intra-individual variation for evaluation of inaccuracy. When expressed in coefficient of variation terms, these allowable limits may be applied at a wide range of levels of quantity.

We sought to investigate the effects of measurement errors on the evaluation of biological variations in healthy subjects. To this end, we analyzed the allowable limits of analytical error which can guarantee the reliability of medical decision levels for interpretation of clinical laboratory data. As a conclusion, we suggest that one-half or less of biological intra-individual variations is appropriate as the criterion for an allowable limit of error to be applied in health check-ups, and this value is in agreement with those in previous reports. If this criteria as the marker for intra-laboratory imprecision is met, a given institute should be able to evaluate time series change follow up of individual data. If the reference interval of laboratory data for disease screening is shared by different institutes, the criterion of 1/4 or less of a biological inter pulse intra-individual variation should be appropriate. This criterion appears to be the goal to be achieved for analytical inter-laboratory variations. On the other hand, as for the criterion for measurement errors which guarantees disease identification based on the cut-off value, a criterion of 1/4 or less of biological inter-pulse intra-individual variation appears to be appropriate, taking into consideration measurement errors which did not influence false-positive or false-negative rates of disease identification. The value turned out to be the same as the limit for the screening of disease. In this study, we considered allowable limits of error in the vicinity of reference value concentrations. However, it will be necessary to set separately the criteria for data in abnormal ranges.

The usefulness of laboratory tests has been assessed by indices such as diagnostic sensitivity, specificity and predictive values. This paper describes the means by which usefulness is evaluated by analysis of prognostic factors, including risk factors and clinical outcome of therapy. It is essential to follow appropriate research designs and to make use of appropriate statistical analyses. Some of the efficient means for analyzing prognostic values include prospective investigation, as represented by cohort studies which follow characteristics of groups for a period of time, and retrospective investigation, as represented by case-controlled studies which evaluate characteristics of groups in a retrospective manner. The appropriate method of analysis for sample data which contain time factors and a relationship between cause and outcome may be multiple logistic regression analysis. This analytical method can present the effects of each factor on a given disease by odds ratios, and can also calculate the occurrence probability of the disease for each case. For example, we analyzed the prognostic factors that govern the effects of interferon therapy on hepatitis type C. We found that the genetic type and quantity of the virus are the major prognostic factors determining the efficacy of interferon therapy, which is in good accord with previous studies. Furthermore, we were able to identify some factors which have not been recognized before. Thus, appropriate research designs not only provide new insights into therapeutic approaches but can also improve the usefulness of laboratory tests.

Sphingosine 1-phosphate (Sph-1-P) is now regarded as a lysophospholipid mediator which is present in plasma and other body fluids, and exerts potent and pleiotropic biological effects [1,2]. Such extracellular mediator activities of Sph-1-P are mainly mediated by subfamilies of G proteincoupled receptors, of which the most completely characterized are those encoded by the endothelial differentiation genes (EDGs) [2,3]. Previously, we showed that in platelets, Sph-1-P is rapidly formed from sphingosine (Sph) by the action of Sph kinase, abundantly stored intracellularly, and released into the extracellular environment upon stimulation [1,4]. Furthermore, vascular endothelial cells express the EDG family Sph-1-P receptors, i.e., EDG-1 and EDG-3 [5]. Consistent with the expression of the high-affinity Sph-1-P receptors in these cells, nanomolar Sph-1-P reportedly induces a variety of vascular endothelial cell responses [5–9]. Sph-1-P stimulates endothelial cell survival or proliferation through a Gi-coupled receptor, probably EDG-1 [5,7]. Furthermore, Sph-1-P induces adherens junction assembly, migration, capillary tube formation, and promotion of angiogenesis [5,8,9]. In this case, the signals mediated via EDG-1 (coupled with Gi) and EDG-3 (coupled with G13 and Gq) are both necessary, and the small GTPase Rhoand Rac-mediated signalings are also involved [5,8]. Very recently, Sph-1-P has been shown to activate endothelial nitric oxide synthase through a novel mechanism involving Akt activation and produce nitric oxide, which plays an important role in endothelial survival and maintenance of the endothelial function [10]. Accordingly, Sph-1-P should be added to the list of platelet-derived bioactive mediators and may play an important role in platelet–endothelial cell interactions, which constitute a central part of vascular biology. To obtain insight into the physiological and pathophysiological role(s) of Sph-1-P in the vascular system, information about the regulation of endothelial cell expression of EDG family Sph-1-P receptors, i.e., EDG-1 and EDG-3, is very important. In fact, the EDG-1 cDNA was originally isolated as a phorbol ester-inducible immediate early transcript from vascular endothelial cells; morphogenetic differentiation of vascular endothelial cells into capillary-like tubules was induced under the conditions [11]. Furthermore, recent data have shown that the mechanical force associated with blood flow, i.e., the fluid shear stress, modulates vascular structure and function, and plays an important role in the pathogenesis of vascular diseases, including atherosclerosis, hypertension, and restenosis [12]. Shear stress has been reported to affect endothelial cell gene expression, and EDG-1 has been cloned as one of two cDNAs encoding a G protein-coupled receptor from a cDNA library of human umbilical vein endothelial cells (HUVECs) exposed to fluid shear stress; EDG-1 mRNA levels increase markedly in response to fluid flow [13]. Accordingly, the modulation of Sph-1-P/EDG signaling may be involved in important vascular responses related to angiogenesis and blood flow conditions. In this study, we examined the possibility that EDG expression, and hence, responsiveness to Sph-1-P in vascular endothelial cells, may be modulated by tumor necrosis factor-a (TNF-a), which is a pleiotropic cytokine that

Elevated Expression of Mitochondrial NADH Dehydrogenase Subunit 4/4L Genes in Vascular Endothelial Cells Undergoing Sphingosine-induced Apoptosis

Biochemical and genetic studies on the arginine-requiring auxotrophs derived from a Serratia marcescens strain were carried out. The arg mutants were classified into seven biochemical groups based on their growth response to five precursors of arginine biosynthesis and enzyme deficiency. Reciprocal transduction tests among these arg mutants divided them into three linkage groups, and the fine mapping in each of the groups by two- or three-point crosses revealed the following arrangement of loci. (1) arg44-thy11-lys1; (2) met1-glt2-argE-(arg19-arg51)-arg120-argG-argH; (3) arg33-pyr4. Five of the seven biochemically distinct arg mutants belonged to the second linkage group, and they constituted an agr-gene cluster. A characteristic feature of the arg-gene cluster of S. marcescens is that it involves argG, which was previously reported only in the Proteus group of Enterobacteriaceae.

Transductional analysis of the chromosomal linkage map ofSerratia marcescensnearleurevealed the following order of loci:ser4-thr3-pyr1-pdx2-leu1-azi8.

A temperate phage, phage PS20, which originated in a lysogenic strain of Serratia marcescens was established to be able to transduce various auxotrophic markers in S. marcescens strains at an equal frequency which ranged from 10–6 to 10–7 per infected cell. Electron microscopic observations show that phage PS20 has a head (550 A) hexagonal in outline and a tail (1000 A × 150 A) with a contractile sheath which is attached to the head.