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Biomedical subjects

J S Gronowitz

Publications and source records attributed to J S Gronowitz.

At least 37 records · Page 2Linked to original sources
PubMed1990

Expression, purification, and crystallization of the HIV-1 reverse transcriptase (RT).

The HIV-1 pol gene proteins (protease, reverse transcriptase, and endonuclease) were expressed in Escherichia coli N4830-1 by the use of the inducible expression vector pWS60 into which the pol gene was inserted. The p66/p51 heterodimer of reverse transcriptase (RT) was isolated in a highly pure and active form. Crystals of the p66/p51 heterodimer were obtained by the vapor diffusion hanging drop technique. The present crystal quality is still not adequate for high resolution X-ray investigation.

Crystallization↗
PubMed1990

Improved assays for DNA-polymerizing enzymes by the use of enzymatically synthesized 5-[125I]iodo-2'-deoxyuridine triphosphate, illustrated by direct quantitation of anti-HIV reverse transcriptase antibody and by serum DNA polymerase analyses.

A one-step procedure which uses enzymes in a crude extract of herpes simplex virus (HSV) type 1-infected cells to synthesize 5-[125I]iodo-2'-deoxyuridine triphosphate [( 125I]dUTP) from [125I]dU is described. The design of a one-step procedure for the purification of the product is also presented. The recovery of [125I]dUTP from [125I]dU varied between 50 and 75%, the radiochemical purity of the product was greater than 90%, and both synthesis and purification were completed within 8 h. The sensitivity and specificity of [125I]dUTP as a substrate for both DNA-dependent DNA polymerase (DNAp) and RNA-dependent DNA polymerase (reverse transcriptase, RT) were evaluated and compared to those of [3H]dTTP for the following specimens: purified cloned Klenow fragment, crude extracts of HeLa-, BHK-, and HSV-2-infected BHK cells, purified avian myeloblastosis virus RT, and purified cloned human immunodeficiency virus (HIV) RT. The [125I]dUTP was accepted as a substrate equally as well [3H]dTTP by all of the specimens at all of the concentrations tested. When the same amount of radiolabel was used, [125I]dUTP gave a sensitivity 10- to 25-fold higher than that of [3H]dTTP. The gain in sensitivity was due to the higher specific activity and a higher counting efficiency of the 125I-label compound. The use of [125I]dUTP also offered technical advantages over alternative substrates available, such as product separation without acid precipitation and exclusion of the need for scintillation cocktails. The half-life of the nucleic also gives a reasonable shelf-life for use in routine assays. Activity of less than 0.3 pg of HIV RT could be detected when the new substrate was used, and this made it possible to quantitate HIV RT antibodies (abs) in diluted serum samples without purifying the immunoglobulin. Analysis of 31 HIV-infected individuals showed that all of them had anti-HIV RT ab and that the amount of serum needed for 50% inhibition of the HIV RT activity corresponded to an amount of immunoglobulin 100-fold smaller (i.e., 0.02-31.4 micrograms) than has been previously reported. With the substrate it was also possible to detect DNAp activity in serum from healthy individuals, although a long-duration assay was required. In a long-duration assay the DNAp activity found in sera from healthy individuals was linear with respect to time, whereas the DNAp activity found in many sera from tumor patients was not. [125I]dUTP is judged to be an excellent substrate for detecting and quantifying the activity of various DNA-synthesizing enzymes and their blocking abs.

Antibodies, Viral↗
PubMed1989

Antibodies against varicella-zoster virus and herpes simplex virus deoxythymidine kinase in heterologous infections.

Antibody responses to varicella-zoster virus (VZV) deoxythymidine kinase (dTK) and herpes simplex virus (HSV) dTK in homologous and heterologous infections were studied. Antibodies blocking the enzymatic activity of VZV-dTK appeared late after varicella and decreased more or less in parallel with the decreasing complement fixing [CF] titre. In herpes zoster, on the other hand, antibodies to VZV-dTK appeared soon after infection. Antibodies against HSV dTKs appeared long after primary infection, but they were subsequently present in all other HSV-CF positive sera. In recurrent HSV, all acute sera were already HSV-dTK antibody positive, and three of nine persons showed an increase in titer between their acute and convalescent sera. Blocking antibodies to VZV-dTK appeared rapidly in specimens from three of 18 individuals positive by an immunofluorescence VZV-immunity test during HSV infection, whereas all other specimens remained devoid of blocking antibodies against VZV-dTK. A rise in antibody titre against HSV-dTK during VZV infections was observed in serum specimens from three of 13 HSV-CF positive patients, whereas an antibody response against HSV-dTK was not found in HSV-CF negative individuals in connection with VZV infections. The relevance of the sporadic increase in the titres of antibodies against heterologous viral dTKs is discussed.

Adolescent↗
PubMed1989

Detection and characteristics of DNA polymerase activity in serum from patients with malignant, viral, or B12-deficiency disease.

DNA polymerase activity was demonstrated in sera from patients with diseases affecting DNA metabolism in different ways, i.e. malignant, viral and vitamin B12-deficiency disease. Using the current procedure, such activity was only detected in sera with pathological levels of thymidine kinase, i.e. no reference level of DNA polymerase activity in healthy individuals could be established. The activity detected for all three types of disease was similar to that of proliferation-associated DNA polymerase alpha, both with respect to sensitivity to different chemical inhibitors and to inhibition by monoclonal antibody. The levels of activity of DNA polymerase and thymidine kinase showed a wide variation and were not significantly correlated when all DNA polymerase-positive sera were included in the analysis. The variation in the ratio of polymerase to kinase activity within a given disease was smaller and the distributions of the enzyme ratios induced by the three types of disease differed significantly. Considering that DNA polymerase activity can be quantitated directly in crude sera, and that such analyses seems to give biological and clinical information, the development of an assay with improved sensitivity for extensive studies is justified.

Biomarkers↗
PubMed1989

Varicella zoster virus deoxythymidine kinase is present in serum before the onset of varicella.

A sensitive enzyme assay with 125I-iododeoxyuridine as substrate and cytidine triphosphate as phosphate donor was used for the direct detection of varicella zoster virus (VZV) deoxythymidine kinase (TK) in human serum. Sera sampled during the incubation period of varicella from 2 patients, a 42-year-old man and his 11-year-old son, have been analysed for TK activity. A simultaneous increase in cellular and VZV TK activity, starting 5 to 3 days before the onset of clinical varicella, was observed.

Adult↗
PubMed1988

Biochemical markers in multiple myeloma: a multivariate analysis.

The analysis of individual biochemical and clinical variables in 121 patients with multiple myeloma showed that serum beta 2-microglobulin (S-beta 2m) had the most significant relation to survival. Other variables such as serum thymidine kinase (S-TK), serum lactate dehydrogenase (S-LDH), S-creatinine, haemoglobin (Hb), ESR, S-albumin, age and clinical stage were also significant. No such relationship was found with M-component, presence of light chains in urine, type of secreted immunoglobulin or S-calcium. The exclusion of clinical stage in the first multivariate analysis resulted in a model consisting of S-beta 2m, age and S-TK, none of the other variables gave additional information. When in the second multivariate analysis the basic variables involved in staging procedure were excluded and clinical stage included, stage III, but not stage II, was found to give additional information to the model described above. Individual analysis of the variables showed that Hb had the most significant relation to effect of initial therapy. Other significant variables were S-TK, S-beta 2m and age. When using the multivariate approach, Hb alone was found to contain all the relevant information.

Adult↗
PubMed1987

An optimized thymidylate kinase assay, based on enzymatically synthesized 5-[125I]iododeoxyuridine monophosphate and its application to an immunological study of herpes simplex virus thymidine-thymidylate kinases.

The biological synthesis and purification of 5-[125I]iododeoxyuridine monophosphate (IdUMP) are described. The specificity of IdUMP as substrate in the thymidylate monophosphate kinase (TMPK) assay is demonstrated, and a 100-fold gain in sensitivity as compared to the conventional TMPK assay is shown. TMPK measurements of isozymes derived from herpes simplex virus (HSV)-infected cells, uninfected cells, and tumor biopsies were performed. The results showed a significant difference in dependence of phosphate donor concentration present for TMPK activity from HSV-infected cells compared to the corresponding activity from uninfected cells, while only a minor difference in pH optima was observed for these enzyme activities. The increased sensitivity made it possible to detect and quantify HSV TMPK-blocking antibodies (ab) present in human sera. Sera from HSV ab-positive individuals were found to block the two HSV TMPKs to varying degrees and with different specificities. The immunological relationship between the TMPK and thymidine kinase (TK) induced by HSV-1 and HSV-2, respectively, was studied by comparing the capacities of different sera to block the two enzymatic activities. The results showed that the capacity to block HSV-1 TK and TMPK was proportional for all of the sera studied, while sera that preferentially blocked only the HSV-2 TMPK or HSV-2 TK were found. It was concluded that the HSV-2 TMPK and TK activities are less related than the corresponding activities for HSV-1 and that the HSV-2 enzyme activities are mediated by different catalytic sites.

Adenosine Triphosphate↗
PubMed1987

Serum deoxythymidine kinase correlates with peripheral lymphocyte thymidine uptake in chronic lymphocytic leukemia.

The serum thymidine kinase (S-TK) and proliferative activity of the leukemic cells were determined in 27 untreated patients with chronic lymphocytic leukemia (CLL). A significant positive correlation between S-TK and proliferation expressed as a proliferative index (PI) was found (r = 0.70, p less than 0.001). Additionally, PI (r = 0.59, p less than 0.01) and S-TK (r = 0.47, p less than 0.05) correlated to peripheral blood lymphocyte count. When different variables and combinations of variables were studied in order to define their capacity for discriminating between progressive and indolent CLL, S-TK activity and PI proved to be powerful indications. In longitudinal studies, both S-TK and PI paralleled disease activity. A model where a combination of S-TK and PI gives information of the degree of localized disease is proposed.

Adult↗
PubMed1986

Serum deoxythymidine kinase in small cell carcinoma of the lung. Relation to clinical features, prognosis, and other biochemical markers.

Thymidine kinase (s-TK), lactate dehydrogenase (LDH), and carcinoembryonic antigen (CEA) were determined in pretreatment serum from 125 patients with small cell carcinoma of the lung. The distribution of marker levels into three ranges, when including all patients were as follows: s-TK less than 5 units 49%, 5-less than 10 units 25%, greater than or equal to 10 units 26%; LDH less than 6.7 mukat 31%, 6.7-less than 13.4 mukat 48%, greater than or equal to 13.4 mukat 21%; CEA less than 7.5 micrograms/l 51%, 7.5-less than 15 micrograms/l 25%, greater than or equal to 15 micrograms/l 24%. The percentages of patients with limited and with extensive disease within each range were s-TK less than 5 82/18, 5-less than 10 29/71, greater than or equal to 10 9/91; LDH less than 6.7 76/24, 6.7-less than 13.4 51/49, greater than or equal to 13.4 21/79; CEA less than 7.5 70/30, 7.5-less than 15 39/61, greater than or equal to 15 23/77. Analyses in relation to metastases present showed that patients with skeletal and bone marrow metastases had significantly higher s-TK and LDH than those without, while this was not the case for CEA. A strong correlation between s-TK and LDH level, a weaker correlation between CEA and s-TK, and no correlation between CEA and LDH level, was found. Both the level of s-TK and LDH correlated to the patients' performance, as defined by the Karnofsky index. These correlations were mainly confined to the patients with extensive disease. Analyses of the prognostic capacity of variables showed that s-TK, stage, and Karnofsky index could divide the patients into groups with highly significant difference in survival time, while LDH and CEA were of less value. Longitudinal studies showed that the serum markers mirrored the disease activity, with the exception that highly increased s-TK was found during remission induction for some patients. It was concluded that the expression of pathologic levels for the serum markers were dependent on different biological parameters. Of the serum markers, only s-TK was judged useful for estimation of disease spread and prognosis of the individual patient.

Actuarial Analysis↗
PubMed1986

Mothers of children with Down syndrome have higher herpes simplex virus type 2 (HSV-2) antibody levels.

The antibody response to herpes simplex virus (HSV) was studied in 53 mothers of children with Down syndrome (Ds) and compared with that in 154 controls, using sera sampled during pregnancy or at delivery. Conventional analysis of HSV complement fixing antibodies showed the same frequency of positivity for the two groups (70%). When the levels of IgG antibodies to an HSV-1 and an HSV-2 antigen preparation were determined by an enzyme-linked immunosorbent assay (ELISA) technique, it was found that the Ds and control mothers had similar levels of IgG antibodies to HSV-1, whereas the level of IgG antibodies to HSV-2 was significantly (P less than 0.001) higher in Ds mothers. The ratio of HSV-2 to HSV-1 ELISA IgG was calculated for each mother and the distribution of these ratios also differed significantly between the control and Ds mothers. The differences found were not due to differences in age distribution in the control and Ds groups. For comparison a third procedure, measurement of thymidine kinase blocking antibody (TK ab), was used. With this procedure the mothers were divided into groups estimated to be positive for HSV-1, HSV-2, or both. Statistical analyses showed a good correlation between the type found in TK ab analyses and the ratio found in the ELISA HSV test. The results clearly demonstrated an overrepresentation of HSV-2 antibody positivity among Ds mothers, though not of sufficient magnitude to imply that HSV-2 can be the major cause of Ds. It is discussed whether HSV-2 might be related to the recently increased birthrate of children with Ds among young mothers in Sweden or to localized geographical clustering of Ds births, or whether the increased HSV-2 antibody positivity merely indicates that factors following the same epidemiological pattern are involved in the aetiology of Ds.

Adolescent↗
PubMed1986

Deoxythymidine kinase, a possible marker for monitoring activity of cytomegalovirus infection after renal transplantation.

Measurement of deoxythymidine kinase activity (S-TK) in serum is used as a marker for cytomegalovirus (CMV) infection following transplant surgery. A case is presented where a kidney transplant patient suffered a fatal CMV infection. The new antiviral drug Foscarnet was used to treat the infection. Retrospective analysis of S-TK showed a good correlation with the course of the disease, its treatment, recurrence and final outcome.

Antiviral Agents↗
PubMed1986

Serum thymidine kinase in transplant patients: its relation to cytomegalovirus activity, renal transplant rejection and its use for monitoring of antiviral therapy.

Twenty patients, cadaveric renal transplant recipients, were retrospectively analysed for serum levels of deoxythymidine kinase. Special reference was made to the thymidine kinase level in relation to rejection, and viral infection. Seven of the patients experienced clinically suspected cytomegalovirus infection. All these patients had elevated levels of serum thymidine kinase during the period of clinical disease. Usually the thymidine kinase level parallelled the severity of the disease. All patients with irreversible rejection had increased levels of serum thymidine kinase, but normally not as high, as seen in patients with severe cytomegalovirus infection. There was also some correlation between clinically suspected rejection, that lead to rejection treatment, and moderate increase in thymidine kinase. However, not all rejection episodes were accompanied by a thymidine kinase increase. Serum thymidine kinase was analysed and compared in patients with self-healing cytomegalovirus disease, and those treated with phosphonoformate. A rapid decline in thymidine kinase level was found in connection with successful antiviral therapy, when compared to the decline in untreated patients. Some bone marrow transplanted patients were also included in this analysis.

Adult↗
PubMed1985

Evaluation of serum deoxythymidine kinase as a marker in multiple myeloma.

A recently developed deoxythymidine kinase assay utilizing 125I-iododeoxyuridine as substrate was used in an investigation of sera from 122 untreated patients with multiple myeloma. Most patients had slightly elevated or normal serum deoxythymidine kinase activity (S-TK), although in some patients values increased by more than forty-fold were found. S-TK correlated with the haemoglobin level but did not correlate with sex, age, erythrocyte sedimentation rate, nor with the serum concentrations of creatinine, beta 2-micro-globulin, Ca or M-component. The distribution of S-TK values in IgG, IgA and pure Bence-Jones myeloma did not differ significantly. Patients with IgG and IgA myeloma excreting light-chain immunoglobulin in the urine had significantly higher S-TK than non-excreters. There was a significant correlation between S-TK values and tumour cell mass as determined by clinical staging. A high pretreatment S-TK (greater than 5.1 units) also distinguished a group of patients with a significantly shorter survival time. Patients with no response to initial therapy had significantly higher S-TK values than those who did respond. In longitudinal studies of 11 patients, S-TK was found to increase when the disease became more aggressive. The possibility of diagnosing disease progression at an early stage by an elevation of S-TK is discussed.

Adult↗
PubMed1985

Thymidine kinase in brain-tumor cysts.

A recently developed method for deoxythymidine kinase (TK) determination was applied to brain-tumor cyst fluid and fluid from a non-neoplastic intracerebral cyst. The fluid from all tumors tested positive for TK whereas the non-neoplastic cyst lacked TK. Cyst fluid was also analyzed for TK before and after intracystic instillation of BCNU. It is suggested that TK activity in the fluid in cystic brain lesions could prove useful in deciding whether an intracerebral lesion is neoplastic. Also, TK activity can be used to evaluate the effect of topical therapy.

Brain Neoplasms↗
PubMed1984

Serum deoxythymidine kinase gives prognostic information in chronic lymphocytic leukemia.

A recently developed deoxythymidine kinase assay, utilizing iodine-125-iodo-deoxyuridine as substrate and capable of detecting enzyme activity in serum from healthy humans, was used in an investigation of sera from 55 untreated patients with chronic lymphocytic leukemia (CLL). When confined to the study, the patients were classified as having progressive or indolent disease and according to Rai stage. The results showed a significant correlation between serum deoxythymidine kinase activity (S-TK) and disease status, i.e., higher values were found in patients with progressive disease, compared to those with indolent disease. S-TK also correlated with Rai stage. S-TK values of more than 40 times the normal value were found in some patients. All patients with S-TK greater than 8.4 units had a disease that was or became progressive during the observation period. Within the patient group with indolent disease two groups that differed with regard to prognosis could be distinguished according to their initial S-TK values. In longitudinal studies of 18 patients with indolent disease, S-TK was found to exceed 8.4 units only on one occasion during an observation period of up to 68 months. In patients with indolent disease, a transition to progressive disease was parallelled by an increase in S-TK. Studies of S-TK levels in 18 patients receiving treatment showed that S-TK decreased during successful therapy. S-TK was also found to increase when the disease was reactivated. From these results it is concluded that S-TK could be used as a prognostic marker for the individual CLL patient. Furthermore, S-TK seems to be useful for longitudinal follow-up studies of disease status, both in indolent disease and in progressive disease during treatment.

Adult↗
PubMed1984

Application of an in vitro assay for serum thymidine kinase: results on viral disease and malignancies in humans.

An improved method for the detection of deoxythymidine kinase (TK) in human sera is reported. The method which utilizes 125I-iododeoxyuridine (IdUrd) as a substrate was used to measure TK in sera from patients with different diseases. Sera collected during the acute stage of infectious mononucleosis were found to contain elevated levels of TK, in most cases 10-40 times the normal value. The serum TK activity disappeared gradually and reached a normal level within 4 weeks. Sera from patients with other viral infections contained in most cases normal serum TK levels except in connection with measles, rubella, varicella, herpes simplex virus and cytomegalovirus infections. Additional studies revealed that sera from patients with different types of advanced lymphomas, acute leukemias, chronic granulocytic leukemia and lung cancer of the small-cell type with metastases, contained high TK levels which fluctuated in parallel with alterations in activity of the disease. The TK activity in sera from patients with both mononucleosis and tumor disease was characterized by electrophoresis and by its ability to utilize cytidine triphosphate as the phosphate donor. The results showed that the serum TK has the same properties as the human cytosolar TKI, except in connection with varicella.

Anemia, Pernicious↗