MicroCT Imaging of Heart Valve Tissue in Fluid.

BACKGROUND: Heart valve computational models require high quality geometric input data, commonly obtained using micro-computed tomography. Whether in the open or closed configuration, most studies utilize dry valves, which poses significant challenges including gravitational and surface tension effects along with desiccation induced mechanical changes. OBJECTIVE: These challenges are overcome by scanning in a stress-free configuration in fluid. Utilizing fluid backgrounds however reduces overall contrast due to the similar density of fluid and tissue. METHODS: The work presented here demonstrates imaging of the mitral valve by utilizing an iodine-based staining solution to improve the contrast of valve tissue against a fluid background and investigates the role of stain time and concentration. RESULTS: It is determined that an Olea europaea oil bath with a relatively high concentration, short stain time approach produces high quality imagery suitable for creating accurate 3D renderings. CONCLUSIONS: Micro-CT scanning of heart valves in fluid is shown to be feasible using iodine staining techniques.

Protein expression studies of desmoplakin mutations in cardiomyopathy patients reveal different molecular disease mechanisms.

Mutations in the gene for desmoplakin (DSP) may cause arrhythmogenic right ventricular cardiomyopathy (ARVC) and Carvajal syndrome (CS). Desmoplakin is part of all desmosomes, which are abundantly expressed in both myocardial and epidermal tissue and serve as intercellular mechanical junctions. This study aimed to investigate protein expression in myocardial and epidermal tissue of ARVC and CS patients carrying DSP mutations in order to elucidate potential molecular disease mechanisms. Genetic investigations identified three ARVC patients carrying different heterozygous DSP mutations in addition to a homozygous DSP mutation in a CS patient. The protein expression of DSP in mutation carriers was evaluated in biopsies from myocardial and epidermal tissue by immunohistochemistry. Keratinocyte cultures were established from skin biopsies of mutation carriers and characterized by reverse transcriptase polymerase chain reaction, western blotting, and protein mass spectrometry. The results showed that the mutation carriers had abnormal DSP expression in both myocardial and epidermal tissue. The investigations revealed that the disease mechanisms varied accordingly to the specific types of DSP mutation identified and included haploinsufficiency, dominant-negative effects, or a combination hereof. Furthermore, the results suggest that the keratinocytes cultured from patients are a valuable and easily accessible resource to elucidate the effects of desmosomal gene mutations in humans.

Postmortem genetic testing of the ryanodine receptor 2 (RYR2) gene in a cohort of sudden unexplained death cases.

The aim of this investigation was to identify pathogenic variants of the ryanodine receptor 2 (RYR2) gene in a cohort of persons aged 0-40 years who died of sudden unexpected death syndrome (SUD), including a cohort of infants who died of sudden infant death syndrome (SIDS). We genetically screened 29 of the 105 exons of the RYR2 gene associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) in 74 cases of SUD without reported structural abnormalities of the heart. Cases were selected from the case database at the Institute of Forensic Medicine, and subsequent mutational screening by DNA sequencing was performed to detect variants in DNA samples extracted from blood samples of deceased persons. A total of 7 of the examined 74 cases were heterozygous for a rare sequence variant in the RYR2 gene. We identified five novel missense variants (p.Q486H, p.D1872N, p.G2367R, p.E4213D, and p.H4579Y), one synonymous variant (p.L4767L), and one previously reported missense variant (p.G4315E). Follow-up studies were possible in family members of three probands (p.Q486H, p.D1872N, and p.H4579Y), and clinical examinations were conducted in family members of two of these probands (p.Q486H and p.H4579Y). In conclusion, we identified a higher prevalence of variants in the CPVT-associated gene RYR2 than in a previously reported cohort of SIDS (9.4% vs. 1-2%). Segregation studies show that one variant (p.H4579Y) co-segregates with CPVT and is presumed to be pathogenic.

Operative management of non-elective incisional hernia reduces readmission in a national database.

PURPOSE: The aim of this project was to compare patient characteristics, overall efficacy, and readmission events following operative vs non-operative management modalities of non-elective patients presenting with symptomatic incisional hernias. METHODS: This study is a retrospective study of patients and patient demographics that presented as non-elective hospitalizations with symptomatic incisional hernia. Analysis of patients and characteristics utilized the National Readmission Database from 2010 to Q3 of 2015, delineating patient factors and outcomes following operative or non-operative management of hernias. RESULTS: A total of 14,137 patients met inclusion criteria for our study. The majority of patients were treated operatively rather than non-operatively (79 vs. 21%) on their non-elective admission for incisional hernia. Those undergoing surgical management were younger (56 vs 61 years, p < 0.01), we more often of male gender (69 vs 64%, p < 0.01), and had fewer comorbidities (1.92 vs 2.97, p < 0.01) and chronic conditions (0.45 vs 2.68, p < 0.01). Patients managed operatively had a significantly lower readmission rate when compared to patients managed non-operatively (6.6 vs 14.3%, p < 0.01). However, non-operative management was associated with a shorter length of stay (3 vs 4 days, p < 0.01). Of patients who were initially medically managed and had to be readmitted, a further 61% underwent surgical treatment on their readmission. CONCLUSION: In this nationwide study, patients with non-elective admissions for incisional hernia were mostly managed surgically. Those managed operatively had lower rates of readmission when compared to non-operative management. Initial non-operative management was associated with a shorter length of stay and a lower cost to the patient. The results of this study support operative management of symptomatic incisional hernia.

CDC field triage criteria accurately predicts outcomes in high impact trauma.

BACKGROUND: The precision of emergency medical services (EMS) triage criteria dictates whether an injured patient receives appropriate care. The trauma triage protocol is a decision scheme that groups patients into triage categories of major, moderate and minor. We hypothesized that there is a difference between trauma triage category and injury severity score (ISS). METHODS: This retrospective, observational study was conducted to investigate a difference between trauma triage category and ISS. Bivariate analysis was used to test for differences between the subgroup means. The differences between the group means on each measure were analyzed for direction and statistical significance using ANOVA for continuous variables and chi square tests for categorical variables. Logistic and linear regressions were performed to evaluate factors predicting mortality, ICU length of stay. RESULTS: With respect to trauma triage category, our findings indicate that minor and moderate triage categories are similar with respect to ISS, GCS, ICU LOS, hospital LOS, and mortality. However, after excluding for low impact injuries (falls), differences between the minor and moderate categories were evident when comparing to ISS, GCS, ICU LOS, and hospital LOS. Additionally, after excluding for low impact injures, ISS, ICU LOS, and hospital stay were found to correlate well with trauma triage category. CONCLUSIONS: In this retrospective, observational study significant differences were not seen when comparing ISS with the trauma triage categories of moderate and minor during our initial analysis. However, a difference was found after excluding for low impact injuries. These findings suggest that CDC criteria accurately predicts outcomes in high impact trauma.

Surgical and non-surgical treatment of inguinal hernia during non-elective admissions in the Nationwide Readmissions Database.

INTRODUCTION: Inguinal hernia repair is one of the most common surgical operations, yet the optimal treatment strategy remains undefined. Treatment of symptomatic inguinal hernias include both surgical and non-surgical approaches. The objective of this study was to determine differences in population, readmission rates, and costs between operative and non-operative approaches for patients admitted non-electively for an inguinal hernia in a national dataset. In addition, we sought to define the baseline characteristics of the two groups and identify potential predictive factors in the non-surgically managed subgroup who were readmitted and treated operatively within 90 days of their first visit. METHODS: This study was a retrospective review of data from the Nationwide Readmissions Database (NRD) from 2010 to 2014. Patients above age 18 who were admitted non-electively for a primary diagnosis of inguinal hernia were included. Patients whose length of stay was < 1% or > 95% percentile or died during the initial visit were excluded. Readmissions within 90 days of the initial visit were flagged. Patients were classified according to initial management strategy: operative versus non-operative. Demographic, clinical, and organizational characteristics were compared between the two cohorts. RESULTS: 14,249 patients met inclusion criteria and were operative (n = 8996, 63.13%) and non-operative (n = 5255, 36.88%) cohorts. When comparing the two groups, readmission rate was lower (0.49% for surgical, 1.78% for non-surgical, p < 0.01), mean length of stay (LOS) longer (3.27 [SE = 0.05] days for surgical, 2.76 days [SE = 0.06] for non-surgical, p < 0.01), and mean total cost higher ($9597 for surgical, $7167 for non-surgical, p < 0.01) in surgically treated patients. The non-surgical population was on average older (63.05 years for surgical, 64.52 years for non-surgical, p < 0.01) with more chronic conditions (3.57 for surgical, 4.05 for non-surgical, p < 0.01). Of the patients initially managed non-surgically, 1.78% (n = 91) were readmitted, and of them, 62.63% (n = 57) were readmitted and managed surgically within 90 days of initial admission (i.e., crossed over from watchful waiting to surgical treatment). Average number of chronic conditions (3.79 versus 4.03, p = 0.74), average number of comorbidities (2.26 versus 2.18, p = 0.87), and average total number of ICD-9 discharge codes (7.44 versus 8.23 p = 0.54 did not differ significantly between the operative versus non-operative sample of the readmitted population. The total cost ($5562.38 versus $8737.28, p = 0.01) was greater in the operative versus non-operative sample. CONCLUSION: Watchful-waiting strategy is the most common treatment approach in patients admitted non-electively for symptomatic inguinal hernia. Readmission after non-elective hospitalization for inguinal hernia is rare, but surgical intervention decreased the likelihood of readmission compared to non-operative management, while also increasing LOS and cost of care. Our data supports a patient centric approach to the management; non-surgical treatment is a viable temporary option even in symptomatic inguinal hernias, while surgical treatment may reduce the likelihood of future readmission.

Hyponatremia as a prognostic and predictive factor in metastatic renal cell carcinoma.

BACKGROUND: Low serum sodium has recently been associated with poor survival in localised renal cell carcinoma (RCC). We now show the prognostic effect of serum sodium in patients with metastatic RCC (mRCC). METHODS: Cohort A comprised 120 consecutive patients with mRCC receiving subcutaneous, low-dose interleukin-2 and interferon-alpha. Hyponatremia was assessed in univariate and multivariate analyses. An independent cohort of another 120 patients with mRCC was used for validation (cohort B). RESULTS: In cohort A, estimated 5-year survival was 15% and median survival was 15.1 months. Serum sodium ranged between 126 and 144 mM. Twenty-four patients (20%) had serum sodium levels below normal range (<136 mM). In multivariate analysis, significant independent risk factors for short survival were low serum sodium (P=0.014), high neutrophils (P=0.018), lactate dehydrogenase >1.5 upper normal level (P=0.002), and number of metastatic sites (+3) (P=0.003). In cohort B, serum sodium ranged between 128 and 146 mM. Seventeen patients (14%) had sodium levels below normal range. In multivariate analysis, serum sodium was validated as an independent prognostic factor (P=0.001). A significant association between lack of response and hyponatremia was observed in both cohorts (P=0.003 and P=0.02, respectively). CONCLUSION: Low serum sodium is a new, validated, independent prognostic, and predictive factor in patients with mRCC.

Hospitalized acute patients with fever and severe infection have lower mortality than patients with hypo- or normothermia: a follow-up study.

OBJECTIVES: Severe infection is a frequent cause of admission to an acute medical unit (AMU). However, not all infected patients present with fever. The aim was to assess differences in 30-day mortality among patients hospitalized with community-acquired severe infection presenting with hypothermia, normothermia or fever. METHODS: A retrospective single-center follow-up at an AMU from August 1, 2009 to August 31, 2011. Patients were included the first time they presented with severe infection within the study period. Temperature was categorized into hypothermia (<36.0ºC), normothermia (36.0ºC-38.0ºC) and fever (>38.0ºC). Severe infection was defined as a discharge diagnosis indicating infection combined with organ failure within the first 24 h after arrival. Multivariable Cox regression analysis was computed to assess the association between temperature and 30-day mortality. RESULTS: A total of 2128 patients with severe infection were included. 3.0% (N = 64) were hypothermic, 57.1% (N = 1216) normothermic and 39.9% (N = 848) had fever at arrival. Crude 30-day mortality was 16.1% (N = 342, 95%CI 14.5-17.7%); 37.5% (N = 24, 95% CI 25.7-50.5%) for hypothermic patients, 18.3% (N = 223, 95%CI 16.2-20.6%) for normothermic patients and 11.2% (N = 95, 95%CI 9.2-13.5%) for patients with fever. Compared to normothermic patients, the adjusted hazard ratio of 30-day mortality among hypothermic patients was 1.62 (95%CI 1.06-2.49) and 0.74 (95%CI 0.58-0.94) among patients with fever. CONCLUSIONS: Over half of the patients admitted to an AMU with severe infection were normothermic at arrival. Hypothermia was associated with an increased risk of short-term mortality, whereas patients with fever were associated with a lower risk compared to those with normothermia.

Identification of a novel mutation in exon 13 of the LDL receptor gene causing familial hypercholesterolemia in two Spanish families.

DNA from 30 unrelated Spanish patients with familial hypercholesterolemia (FH) was studied by single-strand conformation polymorphisms (SSCP)/heteroduplex analysis for mutation detection in exon 13 of low density lipoprotein (LDL) receptor gene. Two patients were found to have an abnormal pattern by heteroduplex analysis, and direct sequencing revealed a C to G substitution at nucleotide position 1965, that results in a Phe to Leu change in codon 634, F634L. We have developed a PCR based assay to detect this mutation in family members. We found three additional F634L mutation carriers, and all of them had high cholesterol levels. Haplotype analysis revealed that all F634L mutation carriers had the same allele determined by TaqI -, StuI +, AvaII +, NcoI -, suggesting the presence of a common ancestor. We report a novel mutation located in exon 13 of the LDL receptor gene that causes FH. We also demonstrate the importance of combining SSCP and heteroduplex analysis to improve mutation detection.

Patients' attitudes toward detection of heterozygous familial hypercholesterolemia.

BACKGROUND: Molecular biology has improved the diagnostic abilities of physicians and enabled them to identify apparently healthy persons with a high risk of genetic disease. OBJECTIVE: To examine the attitudes toward detection of disease and the present well-being in persons at risk of disease with a modifiable outcome, in this case heterozygous familial hypercholesterolemia (FH) and their hypercholesterolemic relatives. MATERIALS AND METHODS: A questionnaire was developed that collected information on the impact on well-being at present and at the time of diagnosis of hypercholesterolemia and on attitudes toward screening family members for heterozygous FH. It was mailed to 62 index patients with heterozygous FH, which was defined by using clinical criteria, and 108 hypercholesterolemic relatives. The response rate was 88%. Results were related to demographic data, experience of psychological or physiologic reactions in relation to awareness of hypercholesterolemia, cardiovascular symptoms, lipid-lowering drug treatment, and information on the DNA-based diagnosis. RESULTS: Of the respondents, anxiety was expressed by 44%, fear of coronary heart disease by 37%, and diminished well-being by 13%. These findings were most pronounced in persons who had experienced physiologic or psychological reactions at the time of diagnosis of hypercholesterolemia or who already had heart disease. Six percent regretted that they were aware of their disease diagnosis, and 84% were in favor of screening for affected individuals in families with a history of heterozygous FH. CONCLUSIONS: The results indicated that a substantial proportion of persons with heterozygous FH had some degree of anxiety. A small minority regretted that they were informed of the diagnosis of heterozygous FH, however, and a majority were in favor of family screening for heterozygous FH.

A sequence variation: 713-8delC in the transforming growth factor-beta 1 gene has higher prevalence in osteoporotic women than in normal women and is associated with very low bone mass in osteoporotic women and increased bone turnover in both osteoporotic and normal women.

Bone mass is partly genetically determined. The genes involved are, however, still largely unknown. Transforming growth factor-beta 1 (TGF-beta 1) is considered a putative regulator of osteoclastic-osteoblastic interaction (coupling). The aim of the present study was therefore to examine whether possible variants of the TGF-beta 1 gene are related to bone mass and osteoporosis. We examined 161 osteoporotic women (at least one low energy spinal fracture) and 131 normal women. We investigated sequence variations in the TGF-beta 1 gene using the single-stranded conformation polymorphism (SSCP) technique combined with DNA sequencing. Seven patients were heterozygous for a cytosine to thymidine base substitution at position 76 in exon 5 (C788-T) (corresponding to position 788 in the TGF-beta 1 cDNA), resulting in a threonine to isoleucine amino acid shift at position 263 in the TGF-beta 1 propeptide (Thr263-Ile). Ten other patients had a one base deletion in the intron sequence 8 bases prior to exon 5 (713-8delC), which could influence splicing. Five normal women exhibited the C788-T sequence variant, and two the 713-8delC. The prevalence of 713-8delC was significantly higher in the osteoporotic group (chi 2 = 4.02, p < 0.05). Osteoporotic patients with the 713-8delC variant had increased levels of bone alkaline phosphatase (p < 0.05). If the osteoporotic patients with a z score of the lumbar spine below -1 were examined separately, we found increased serum levels of bone alkaline phosphatase (p < 0.05), increased urinary excretion of hydroxyproline (p < 0.05), and reduced bone mass of the lumbar spine (p < 0.05) in patients with 713-8delC. No correlation to bone mass was demonstrated in the normal women, but 713-8delC was associated with increased serum levels of bone alkaline phosphatase (p < 0.05). The sequence variation, 713-8delC, in the TGF-beta 1 gene is more frequent in patients with osteoporosis compared to normal controls. The 713-8delC variant seems to be associated with very low bone mass in osteoporotic women with low bone mass and increased bone turnover in both osteoporotic and normal women.

The Trp23-Stop and Trp66-Gly mutations in the LDL receptor gene: common causes of familial hypercholesterolemia in Denmark.

Mutations in the gene for the low density lipoprotein (LDL) receptor cause the autosomal dominant disease familial hypercholesterolemia (FH), the prevalence of which is about 0.2% in most populations. By PCR-SSCP analysis and direct sequencing, we identified the receptor-negative Trp23-Stop LDL receptor mutation (FH Cincinnati-5) in 10 of 63 FH probands and the receptor-defective Trp66-Gly LDL receptor mutation (FH French Canadian-4) in another 10 of the 63 FH probands. These two mutations thus account for 30% of diagnosed FH families in Denmark. Comparison of the mean lipid concentrations (unadjusted and adjusted for age), including serum total cholesterol and LDL-cholesterol, showed no significant differences between the two groups of FH heterozygote probands (cholesterol: 10.7 mmol/l vs. 10.7 mmol/l) and between the probands and 16 and 22 non-proband family members with the Trp23-stop (cholesterol: 10.1 mmol/l) ad Trp66-Gly (cholesterol: 10.7 mmol/l) mutations, respectively.

Spectrum of LDL receptor gene mutations in Denmark: implications for molecular diagnostic strategy in heterozygous familial hypercholesterolemia.

Heterozygous familial hypercholesterolemia (FH) is one of the most common potentially fatal single-gene diseases leading to premature coronary artery disease, but the majority of heterozygous FH patients have not been diagnosed. FH is due to mutations in the gene coding for the low-density lipoprotein (LDL) receptor, and molecular genetic diagnosis may facilitate identification of more FH subjects. The Danish spectrum of 29 different mutations, five of which account for almost half of heterozygous FH, is intermediate between that of countries such as South Africa, where three mutations cause 95% of heterozygous FH in the Afrikaners, and Germany or England, where there are many more mutations. In clinical practice, a strategy for the genetic diagnosis of heterozygous FH, tailored to the mutational spectrum of patients likely to be seen at the particular hospital/region of the country, will be more efficient than screening of the whole LDL receptor gene by techniques such as single-strand conformation polymorphism (SSCP) analysis in every heterozygous FH candidate. In Aarhus, Denmark, we have chosen to examine all heterozygous FH candidates for the five most common LDL receptor gene mutations (W23X, W66G, W556S, 313 + 1G --> A, 1846 - 1G --> A) and the apoB-3500 mutation by rapid restriction fragment analysis. Negative samples are examined for other mutations by SSCP analysis followed by DNA sequencing of the exon indicated by SSCP to contain a mutation. If no point mutation or small insertion/deletion is detected, Southern blot or Long PCR analysis is performed to look for the presence of large gene rearrangements. In conclusion, our data suggest that an efficient molecular diagnostic strategy depends on the composition of common and rare mutations in a population.

P1A1/A2 polymorphism of platelet glycoprotein IIIa and risk of acute coronary syndromes in heterozygous familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is an autosomal inherited disorder caused by different mutations in the low density lipoprotein (LDL) receptor gene. It has been demonstrated that there is an increased risk of coronary heart disease (CHD) in heterozygous FH subjects, although this excess CHD is not only explained by the LDL-cholesterol concentration or the class of the LDL-receptor mutation. To investigate if a common polymorphism at the platelet glycoprotein (GP) IIIa gene locus could be related to CHD phenotypic variation in heterozygous FH. we have carried out a case-control study. We have studied 40 cases and 40 controls matched for age, sex and genetic defect in the LDL-receptor gene. Allele frequency of PI(A2) polymorphism for cases and controls was 20 and 22.5%, respectively, and the difference was not significant. In conclusion, our data do not support any association between the GP IIIa polymorphism and the increased prevalence of acute coronary syndromes in the heterozygous FH subjects.

A common W556S mutation in the LDL receptor gene of Danish patients with familial hypercholesterolemia encodes a transport-defective protein.

In a group of unrelated Danish patients with familial hypercholesterolemia (FH) we recently reported two common low-density lipoprotein (LDL) receptor mutations, W23X and W66G, accounting for 30% of the cases. In this study, we describe another common LDL receptor mutation, a G to C transition at cDNA position 1730 in exon 12, causing a tryptophan to serine substitution in amino acid position 556 (W556S). In the Danish patients, the W556S mutation was present in 12% of 65 possible mutant alleles. The pathogenicity of the W556S mutation, which is located in one of the five conserved motifs Tyr-Trp-Thr-Asp in the epidermal growth factor homology region, was studied in transfected COS-7 cells expressing normal and mutant LDL receptor cDNAs. Results obtained by immunofluorescence flow cytometry and confocal microscopy, as well as by immunoprecipitation, were compatible with complete retention of the mutant protein in the endoplasmic reticulum. The transport-defective W556S mutation and the W23X and W66G mutations seem to account for about 40% of the LDL receptor defects in Danish families with FH.

Overexpression of human thymidine kinase mRNA without corresponding enzymatic activity in patients with chronic lymphatic leukemia.

The level of cytosolic thymidine kinase (TK1) mRNA in lymphocytes from six healthy people and in lymphocytes from five patients with untreated chronic lymphatic leukemia (CLL) was determined with competitive polymerase chain reaction (competitive PCR). Using this procedure we have shown that in patients with CLL, there is an overexpression of TK1 mRNA without corresponding enzymatic activity. The TK1 mRNA level is approximately 100-fold higher in lymphocytes from CLL patients than in lymphocytes from healthy persons. A high level of TK1 mRNA without corresponding enzyme activity may indicate a defect in the processing of the enzyme. This may disturb the cells' normal feedback system and thereby influence the development of malignant conditions.

Orchitis and testicular abscess formation caused by non-typhoid salmonellosis. A case report.

The case of a 36-year-old man with epididymo-orchitis followed by testicular abscess formation 4 weeks later due to Salmonella berta is reported, and the literature on orchitis and testicular abscesses caused by non-typhoid Salmonella is reviewed.

Flow cytometric assessment of effects of fluvastatin on low-density lipoprotein receptor activity in stimulated T-lymphocytes from patients with heterozygous familial hypercholesterolemia.

To test the effects of fluvastatin on low-density lipoprotein (LDL) receptor activity in patients with heterozygous familial hypercholesterolemia, the authors measured LDL receptor activity in stimulated T-lymphocytes prepared from 34 patients before and after treatment with 40 mg fluvastatin daily for 12 weeks. Maximally induced pretreatment LDL receptor activities did not correlate with pretreatment plasma cholesterol levels or with changes in plasma cholesterol levels during treatment, and there were no significant changes in LDL receptor activity during treatment. Barring methodological problems, two explanations are possible. Insofar that LDL receptor activity in lymphocytes reflects LDL receptor activity in the liver, the results suggest that the primary response to treatment with fluvastatin in heterozygous familial hypercholesterolemia (FH) patients is not enhanced LDL receptor activity. Alternatively, fluvastatin increases LDL receptor activity in hepatocytes but has little effect on receptor-dependent lipoprotein catabolism in extrahepatic tissues in vivo.

Human thymidine kinase 1. Regulation in normal and malignant cells.

In mammalian cells, salvage pathway phosphorylation of thymidine is catalyzed by two thymidine kinases: the cell-cycle regulated cytoplasmic TK1 and the constitutively expressed mitochondrial TK2. Since TK1 is virtually absent in non-dividing cells, TK2 is probably the only thymidine kinase present in these cells. In cellular metabolism, TK1 and TK2 presumably serve to maintain sufficient dTTP for DNA replication and repair. TK1 purified from phytohemagglutinin-stimulated human lymphocytes is a dimer in the absence and a tetramer in the presence of ATP. In addition to the molecular weight transition, incubation with ATP at 4 degrees C or storage with ATP induces a reversible, enzyme concentration-dependent, kinetically slow transition from a low to a high affinity form of TK1, with Km values of 14 microM and 0.5 microM, respectively. This affinity difference implies that at cellular thymidine concentrations, the difference in catalytic activity between the two TK1 forms will be 3-5-fold. Calculations of cellular TK1 concentration suggested that the low affinity dimer form was dominant in G0/G1 cells and the high affinity tetramer form in S-phase cells. Hence, the transition may serve to fine-tune the cell-cycle regulation of thymidine kinase activity on the post-translational level. To study the ATP effect on the molecular level, an IPTG inducible T7 RNA polymerase-dependent expression system for the entire human TK1 polypeptide in E. coli was established. The recombinant TK1 has the same subunit mass and specific activity as the native enzyme. However, the recombinant TK1 solely displayed the kinetics of the high affinity form, with Km values of 0.3-0.4 microM regardless of pre-exposure to ATP, indicating that the ATP effect may be dependent on post-translational modifications absent in E. coli. Surprisingly, we did not observe any effect of ATP on TK1 purified from bone-marrow cells from a patient with acute monocytic leukemia (AMOL). Furthermore, the Km values of TK1 from these cells were 45 microM for the ATP-free enzyme and 65 microM for the ATP-incubated enzyme. With TK1 purified from HL-60 cells, we obtained the same pattern and kinetic values as for TK1 from lymphocytes. In the light of the results with the recombinant TK1, we presume that the lack of ATP effect and very high Km values observed for the AMOL TK1 may be due to changes in post-translational regulatory mechanisms in acute monocytic cells.