Biomedical subjects
Publications and source records attributed to M Karp.
The authors report on three infants who had an unusual variant of sacrococcygeal teratoma--a highly vascularized tumor. All three patients had major problems perioperatively, which resulted in cardiac arrest in all three and the death of one. Several suggestions are made concerning diagnosis as well as preoperative and intraoperative management of this rare tumor variant.
The antimicrobial activities of two nitric oxide-releasing compounds against Escherichia coli were investigated by using recombinant E. coli cloned with a luciferase gene from Pyrophorus plagiophthalamus. Since luciferase uses intracellular ATP to generate visible light which can be measured from living cells in real time, we wanted to compare the extent to which cell viability parallels light emission. Results from luminescence measurements and CFU counts were in good agreement, and the decrease in light emission was shown to provide a rapid and more sensitive indication of cytotoxicity.
A prospective survey of all newly diagnosed insulin-dependent diabetes mellitus (IDDM) children and adolescents aged 0-17 years in Israel was conducted for the years 1989 and 1990. All diabetic clinics in Israel treating young diabetics were contacted and they returned written reports to us. Each clinic was also visited regularly by a member of the team who reviewed the individual charts to obtain data on population origin as well as medical and demographic data. A total of 187 patients were identified (164 Jews and 23 Arabs), giving a total incidence rate of 5.46/10(5). Analysis of the incidence rates by population groups showed that Arabs and Jews originating in Asia had the lowest incidence (2.77 and 4.58/10(5) respectively), followed by Jews whose fathers were born in Israel (5.61/10(5)). The highest incidence was registered for Jews originating from Europe and North America (9.34/10(5)). The female-to-male preponderance ratio was higher in the Jews originating in Asia (2.1) than in Jews originating in Europe and North America (1.2). Comparing the present data with a survey performed for the years 1975-80 we found a statistically significant increase in incidence in all population groups. Our findings strongly suggest an influence of genetic factors on the incidence of childhood IDDM.
The cDNA encoding human placental cytochrome P450 aromatase (CYP19) was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression vector system. The recombinant protein product was characterized by Northern and Western blot analyses as well as by direct measurement of aromatase activity. The expressed enzyme proved to be both catalytically active in the presence of P450 reductase and immunologically reactive with polyclonal antibodies raised against human placental aromatase. The activity of aromatase increased 10% after the addition of 0.1 microgram/ml hemin chloride to the culture medium. However, the level of aromatase protein decreased considerably when the concentration of hemin chloride reached 10 micrograms/ml indicating that hemin chloride has toxic effects on the lepidopteran insect cell line. In conclusion, the baculovirus system is suitable for high level expression of functional human placental CYP19.
A click beetle luciferase-based baculovirus expression vector is described for functional analysis and high level expression of a human alpha 2-adrenergic receptor (alpha 2AR) in Sf9 insect cells. The resultant recombinant baculovirus construct, AcLucGR-alpha 2(C4), was isolated by utilizing the light emitting properties of luciferase and used for abundant expression of the alpha 2C-C4 receptor protein in this lepidopteran insect cell line. A maximal expression of alpha 2-receptors at a level of 1.370 pmol/mg protein was obtained at 48 h after infection as determined by ligand-binding experiments using the alpha 2-receptor antagonist, [3H]rauwolscine. The receptor agonists, noradrenaline and clonidine, displaced the [3H]rauwolscine binding with Ki values 12.3 +/- 1.54 microM and 1.23 +/- 0.11 microM, respectively. The recombinant receptors were functionally intact since the agonists inhibited forskolin-stimulated cAMP production. Here, however, the maximal inhibition was obtained at 36 h after the infection. The results presented here, suggest that the baculovirus expression vector system (BEVS) provides a simple method for abundant expression of functional alpha 2-receptor subtypes. In addition, co-expression of luciferase proved to be useful for screening and isolation of the recombinant baculovirus.
The genes encoding the alpha- and beta-chains of the human interleukin-2 receptor were expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding genes were inserted under the polyhedrin promoter of the Autographa california nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line during viral infection. The recombinant receptor proteins were identified in the insect cell lysates by using protein dot blot and ELISA techniques. At 36 h post infection the corresponding proteins were clearly detected using anti-IL-2 alpha- and beta-receptor-specific antibodies. A large amount of the alpha-chain was also found in the supernatant culture media at 72 h post infection and metabolic labelling with [35S]-methionine indicated that it was proteolytically cleaved into a 32 kDa soluble form. A similar soluble or secreted form of the beta-chain was, however, not observed. Both receptor proteins were expressed on the surface of the insect cells as determined by flow cytometry analysis. Studies performed with the different IL-2 receptor forms (alpha- and beta-chains alone or in combination) in the presence or absence of rIL-2 suggest that the receptor proteins when expressed in infected insect cells are non-functional with respect to tyrosine phosphorylation.
A fusion construct encoding two antibody-binding sites of protein A from Staphylococcus aureus and click beetle, Pyrophorus plagiophthalamus, luciferase (LucGR) was designed and expressed using the baculovirus system. The construct was inserted under the transcriptional regulation of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed in the insect Spodoperta frugiperda cell line during viral infection. The properties of the resultant chimeric protein product, protA-LucGR, were studied both in vivo and in vitro by using i) luminometry, ii) immunoblot analysis, iii) immunoprecipitation, iv) metabolic labeling procedures and v) luminescent immunoassays. Together, the results clearly demonstrate that the light-emitting properties of the fused luciferase construct remain intact. Further, the antibody-binding domain of protein A retains its activity as it binds to both rabbit and goat as well as human immunoglobulins. Due to the dual biological function of this fusion protein, it should provide a potential reagent within the field of molecular biology and diagnostics.
A group of vectors for luciferase expression in Bacillus subtilis was constructed. So far, only bacterial luciferases have been expressed in Bacillus, but in this study we wanted also to express genes encoding eukaryotic luciferases to perform direct comparisons of the light levels produced by the two different systems in B. subtilis. The vectors constructed can replicate both in Escherichia coli and B. subtilis, and the luciferase expression is strictly regulated due to the dual plasmid system used. Nearly a 100-fold increase in light production compared to previous results was achieved when genes encoding bacterial luciferase were inserted into the constructs and transformed into B. subtilis. An additional tenfold increase in light production was obtained when luciferase genes from the North American firefly (Photinus pyralis) or a click beetle (Pyrophorus plagiophtalamus) were introduced in a similar fashion into B. subtilis. Measurement of the light emission was performed without disruption of bacterial cells in a real-time manner, which is a common feature when working with all of these constructions. Structures of the shuttle vector constructs and results from light emission measurements are presented.
A retrospective study was undertaken to determine if there exists an association between cryptorchidism and the intra-abdominal wall defects of gastroschisis and omphalocele. The records of 25 newborn male infants (13 with omphalocele, 12 with gastroschisis) were examined. In this sample there was no statistically significant association between these defects and cryptorchidism in either the premature or the full-term infants, when compared with a healthy population. Further clinical studies with larger numbers of patients are recommended.
The management of 26 children with major renal injury secondary to blunt trauma was reviewed. Emergency computerized tomography (CT) was performed in all instances. Injury ranged from parenchymal laceration to vascular avulsion. Early surgical exploration was done in 5 children due to hemodynamic instability, renal pedicle injury or suspected malignancy. The remaining 21 children were observed. Of these children 5 had associated intra-abdominal organ injuries. The average length of hospitalization was 13.4 days and the average intensive care unit stay was 6.9 days. A third of the children were transfused with an average 10.8 cc/kg. of packed red cells. Ten patients (47.6%) had febrile episodes that lasted an average of 3 days. No foci of infection other than bladder urine were identified and there were no infected perirenal collections. In 2 children ureteral stents were placed cystoscopically. Exploration was performed in 1 child for delayed hemorrhage 2 months after hospital discharge. Followup CT was available in 15 patients and all kidneys functioned, including 3 with residual focal scarring, 2 with parenchymal calcifications and 1 with a cyst. Eleven patients were evaluated clinically at least 1 year after injury and all were asymptomatic, while 1 child had mild diastolic hypertension. In conclusion, nonoperative management results in an excellent long-term outcome in the majority of cases. In-hospital morbidity is minimal and early surgical exploration should be reserved for those with hemodynamic instability or renal pedicle injury. Immediate CT is an invaluable aid in categorizing and managing these patients.
We have developed a fast and sensitive system for the in vivo analysis of gene expression in baculovirus infected lepidopteran insect cells. A recombinant baculovirus containing a luciferase gene from the click beetle, Pyrophorus plagiophthalamus, under transcriptional regulation of the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus (AcNPV) was used to infect a Spodoptera frugiperda cell line. Recombinant luciferase could be monitored by luminometry in real-time without disruption of the infected cells, allowing detection of synthesis as early as one hour after infection. The range of luminescence measurements was normally over four orders of magnitude, and the kinetics of luciferase synthesis and the levels of light produced in vivo closely correlated with the expression of polyhedrin in AcNPV infected cells when analyzed by SDS-PAGE. Additionally, single infected cells could be identified by CCD image analysis and flow cytometry.
A total of 614 Jewish patients under the age of 18 with Type 1 (insulin-dependent) diabetes mellitus, diagnosed in Israel during the period 1 January 1965 to 31 December 1979, were identified by exhaustive screening of all possible sources. Mortality experience of this cohort was updated to 31 March 1988 through the Central Population Registry and 14 deaths were identified. The ascertainment rate for diagnosed cases as well as for deaths is estimated at about 95%. There was a significantly higher (p less than 0.001) by 3.2-fold excess mortality relative to the age and sex-adjusted mortality as expected the general Jewish population in Israel. This excess was due to three cause-of-death categories: diabetic ketoacidosis (n = 3; p less than 0.001), cardiovascular diseases (n = 3; p less than 0.001) and infections (n = 2; p = 0.03). The rate of malignancies (n = 2), external causes (n = 3) and other general causes (n = 1) did not differ significantly from that expected. During the first 15 years of the disease cumulative mortality resembled that of the general population, with a subsequent steep increase so that by 20 years disease duration, the rate was four-fold higher than expected. This mortality pattern was similar irrespective of age at onset, sex and ethnic group (Ashkenazi vs non-Ashkenazi Jews). A factor contributing to the lack of increase in mortality rate in the first 15 years of Type 1 diabetes may be the comprehensive multidisciplinary treatment approach employed for most juvenile diabetic patients in Israel leading to early referral and an overall better metabolic control.
Risk factors associated with diabetic microvascular complications, with special reference to ethnic origin, were looked for in 231 young Jewish insulin-dependent diabetes mellitus (IDDM) patients with duration of diabetes greater than or equal to 10 yr. Median age at diagnosis of diabetes was 9.2 yr (range 0.04-26.2 yr), and median duration of the disease was 15.3 yr (range 10.0-37.2 yr). Sixty-three percent of the patients were Ashkenazi Jews, and 37% were non-Ashkenazi Jews. HbA1 was evaluated every 3 mo in the last 10 yr of follow-up, and albumin excretion rate was tested in three 24-h urine collections. Direct and indirect ophthalmoscopy was performed every year since diagnosis of diabetes, and if retinal pathology was suspected, color photographs were taken. Microalbuminuria was detected in 31% and macroalbuminuria in 7% of the patients. Nonproliferative and proliferative retinopathy was found in 44 and 12% of the patients, respectively. On logistic regression analysis, two variables were significantly and independently associated with diabetic nephropathy--non-Ashkenazi origin and mean HbA1 values over the first 5 of 10 yr of follow-up. Variables significantly and independently related to diabetic retinopathy were non-Ashkenazi origin, mean HbA1 values over the last 10 yr of follow-up, and duration of diabetes. Because non-Ashkenazi Jews in Israel are of lower socioeconomic status than Ashkenazi Jews, we stratified our patients according to their socioeconomic parameters, median HbA1 values, and duration of diabetes. Non-Ashkenazi patients were at a higher risk to develop complications in all strata.(ABSTRACT TRUNCATED AT 250 WORDS)
The relationship between structure, activity, and stability of the thermostable Bacillus stearothermophilus alpha-amylase was studied by site-directed mutagenesis of the three most conserved residues. Mutation of His-238 to Asp involved in Ca2+ and substrate binding reduced the specific activity and thermal stability, but did not affect the pH and temperature optima. Replacement of Asp-331 by Glu in the active site caused almost total inactivation. Interestingly, in prolonged incubation this mutant enzyme showed an altered end-product profile by liberating only maltose and maltotriose. Conservative mutation of the conserved Arg-232 by Lys, for which no function has yet been proposed, resulted in lowered specific activity: around 12% of the parental enzyme. This mutant enzyme had a wider pH range but about the same temperature optimum and thermal stability as the wild-type enzyme. Results obtained with different mutants were interpreted by computer aided molecular modeling.
Two-hundred-and-fifty patients with juvenile diabetes mellitus aged 3-38 years, were examined for trigger finger. Thirteen patients (5%) were found to have trigger finger--10 women and 3 men aged 14-38 years (mean 26 years). The ring, middle fingers, and thumb were the most affected. Two patients had bilateral trigger finger. There was a significant correlation between duration of diabetes and trigger finger (p less than 0.001) but no correlation with the control of diabetes. Our work indicates for the first time the prevalence of trigger finger in young patients with insulin dependent diabetes mellitus.
247 patients with juvenile diabetes mellitus, aged 3-37 years, were examined for diabetic hand syndrome. 68 (27%) had 1 or more of the manifestations of diabetic hand syndrome. In 45 (18%) flexion contractures were found, 41 (17%) had skin changes resembling those of scleroderma and digital sclerosis, and 12 (5%) suffered from trigger finger. We found an association between diabetic hand syndrome and diabetes control as evaluated by serial levels of hemoglobin A1c measured during the years of follow-up. A high relative risk for microvascular complications was found in those who had diabetic hand syndrome, compared to the others. The relative risk for retinopathy was 2.5 times greater in patients with diabetic hand syndrome (p less than 0.001). These results show that diabetic hand syndrome is a common presentation of juvenile diabetes mellitus and can be utilized as a marker for some of its complications.
For gene expression and cell physiology studies of Gram-positive Bacillus subtilis, novel shuttle vectors which cause the host organism to produce light have been constructed. These vectors carry genes encoding luciferase from Vibrio harveyi, a selectable kanamycin marker and an origin of replication for Gram-negative and -positive bacteria. The effect of DNA-insert size on light production in Escherichia coli and in B. subtilis was studied by also cloning into the shuttle vector a gene whose product participates in fatty acid metabolism. B. subtilis containing lux genes was found to differ from its Gram-negative counterpart in light emission characteristics. After addition of the substrate, light emission by B. subtilis was rapid but it decayed quickly, showing biphasic kinetics. In E. coli light emission is continuous, reflecting constant regeneration of substrates for the luciferase reaction. A promoter cloning vehicle, pCSS117, was constructed by inserting a transcriptional termination loop in the upstream sequences of luciferase genes. Using this vector, the mode of action of promoters of interest can be studied in E. coli and in B. subtilis.
We have studied possibilities for constructing Escherichia coli strains capable of producing stable light. Light production in E. coli is achieved by cloning the genes encoding bacterial luciferase from Vibrio harveyi. To gain the advantage of sensitive detection of light we transferred the genes under the control of a strong, regulatable promoter system. Stabilization of light produced by E. coli clones was accomplished by finding the optimal plasmid construction and growth conditions as well as suitable measuring buffers. The adjustment of the luciferase synthesis for bioluminescence measurements to a high but not harmful level gives healthy cells and stable luciferase. Cultivation at 30 degrees C in an uninduced state was found to be the most important factor in getting stable-light production. The overall cell metabolism being unstressed gives us the possibility of monitoring cell physiology and factors affecting it via bioluminescence reactions in vivo. To make the results easy to interpret the light emission has to be stable during a measurement period of one to several hours. In the case of the original light-producing bacteria, Vibrio and Photobacterium strains it has not thus far been possible to find conditions where light emission would be stable for several hours. Based on our findings an automated biosensor system can be developed to monitor the effects of biologically active compounds against stable-light-producing bacteria.