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Biomedical subjects

K N Subramanian

Publications and source records attributed to K N Subramanian.

At least 19 recordsLinked to original sources
PubMed1992

Intravenous immune globulin therapy for early-onset sepsis in premature neonates.

Newborn infants may have IgG deficiencies that increase their susceptibility to bacterial infection. To determine whether intravenous immune globulin (IVIG) therapy improves survival rates in early-onset sepsis, we prospectively entered 753 neonates (birth weight 500 to 2000 gm, gestation less than or equal to 34 weeks, age less than or equal to 12 hours) into a multicenter, double-blind, controlled trial. Blood culture specimens were obtained and infants randomly assigned to receive 10 ml (per kilogram) intravenously of a selected IVIG (500 mg/kg) or albumin (5 mg/kg) preparation. Maternal and neonatal risk factors were not different between groups. Thirty-one babies (4.2%) had early-onset sepsis; the causative organisms were group B streptococcus (12 babies), Escherichia coli (6), and others (13). Of these 31 neonates, 7 (23%) died. Total serum IgG was higher for 7 days after IVIG therapy than after albumin treatment (p less than 0.05). During these 7 days, 5 (30%) of 17 albumin-treated and none of 14 IVIG-treated patients died (p less than 0.05). The survival rate at 56 days of age, however, was not significantly improved. Group B streptococcus type-specific IgG antibody was significantly increased after IVIG treatment and appeared to be related to the amount of IVIG specific antibody. Infusion-related adverse reactions were less frequent in patients receiving IVIG therapy (0.5%) than in those receiving albumin. The IVIG therapy in neonates with early-onset sepsis, while reducing the early mortality rate, did not significantly affect the overall survival rate. Further studies are necessary to confirm these findings and to determine more effective therapeutic regimens.

Anti-Bacterial Agents↗
PubMed1991

Sequence requirements for activation of replication by the SV40 transcriptional promoter or enhancer elements.

Previous studies have demonstrated that the 21- or the 72-bp repeat transcriptional control elements enhance the efficiency of SV40 DNA replication in vivo, provided either of these repeats is located near the end of the core replication origin containing the 17-bp A + T-containing sequence. Using two sets of point mutants we have investigated the contributions of the various sequence motifs present in the 21- or the 72-bp repeats toward activation of replication. Regarding the contribution of the six GC motif components of the 21-bp repeats, we find that GC motif I, located closest to the core origin, is dispensable for activation of replication. A mutation in GC-I in fact causes an increase in replication efficiency. We also find that GC motifs I and II present in the nontandem copy of the 21-bp repeats are not sufficient to activate replication. Our present study indicates that a combination of three GC motifs such as II, III, and IV (including one of the two perfect, tandem copies of the 21-bp repeats) is important for activation of replication. Regarding the 72-bp repeat transcriptional enhancer region, we find mutations in a number of its individual motifs to have a negative consequence on replication, with mutations in the GT-I*/TC-II and Sph-II/octamer motifs exhibiting the most negative effects. Overall, we find that the replication activation effects of the 21- and the 72-bp repeats require the participation of multiple motifs present in them. Cellular factors binding to these motifs are expected to mediate their replication activation effects. For the most part, the motifs required for activation of replication are the same as those reported in earlier studies to be important for efficient early and late viral mRNA transcription.

Base Sequence↗
PubMed1991

Efficient replication of plasmids containing the SV40 origin in N-myc overexpressing human neuroblastoma cells.

Using test plasmids containing the SV40 origin, we found a wide spectrum of permissiveness to their replication in different human cell lines. N-myc overexpressing neuroblastoma cells were highly permissive. LA-N-1 neuroblastoma cells were the most permissive of all the cell lines that we tested including the homologous CV-1 or COS-1 monkey kidney cells. Other human cell lines expressing various amounts of c-myc, and the 293 cell line expressing adenovirus E1A and E1B exhibited intermediate levels of permissiveness. T24 and EJ bladder carcinoma cells, which do not express the myc genes, were nonpermissive. Transient expression of c-myc or N-myc from plasmid vectors resulted in a modest stimulation of replication. Replication of test plasmids containing different configurations of the SV40 origin region was activated by the myc proteins. The high efficiency of replication in LA-N-1 cells is due to a combination of reasons including the overproduction of N-myc, high efficiency of expression of the SV40 replication initiator protein large T antigen from a cotransfected expression plasmid (containing the T antigen gene under the RSV LTR control), and other unknown host cell replication stimulatory factors. Replication of test plasmids was not detected in N-myc or c-myc overexpressing cells when the T antigen expression plasmid was not provided, showing that the myc proteins cannot substitute for T antigen in SV40 DNA replication.

Antigens, Viral, Tumor↗
PubMed1989

Pulmonary management during extracorporeal membrane oxygenation.

Traditional lung management during extracorporeal membrane oxygenation (ECMO) calls for low inspiratory and expiratory pressures with low ventilator rate to achieve lung rest. However, rapid weaning of pressures to severely injured lungs commonly leads to marked increase in pulmonary opacification as seen on chest x-ray and loss of volume. We postulated that a sufficiently high level of PEEP could prevent this deterioration. Forty-six newborns, who required ECMO therapy for refractory respiratory failure, were maintained on 8 to 14 cm H2O PEEP. The peak pressure was 20 to 24 cm H2O, rate 10 to 15 breath/min, and FIO2 0.21. Forty-one (89%) of 46 patients survived to discharge. The duration of ECMO was significantly decreased compared to the national average (82.4 +/- 43 vs. 117.5 +/- 59 h). The lungs of most patients remained adequately expanded and free of severe pulmonary opacification. Eleven of 40 patients did show significant worsening of chest x-ray compared to baseline, but only one of 18 who were on PEEP of greater than or equal to 12 cm H2O showed such deterioration. No unexpected complications were encountered. These data suggest that the use of high PEEP during ECMO safely prevents deterioration of lung function and promotes more rapid lung recovery. PEEP levels of 12 to 14 cm H2O appear to be most effective.

Extracorporeal Membrane Oxygenation↗
PubMed1988

Functional anatomy of the simian virus 40 late promoter.

We have examined the control sequences for the late promoter function of simian virus 40 (SV40) in COS-1 cells which produce SV40 T antigen constitutively. Plasmids were constructed by cloning mutant late promoter segments upstream from sequences coding for the bacterial chloramphenicol acetyltransferase (CAT) gene, and were converted to "double-origin" type by inserting functional replication origin segments downstream from the CAT gene for replicative competence when necessary. The late promoter activity was determined by transient expression assay of the CAT mRNA and enzyme activity levels following DNA-mediated gene transfer into COS-1 cells. We find that the minimal replication origin and the 21-bp repeat containing T antigen and transcription factor Sp1 binding sites, respectively, are dispensable for late promoter function provided that one copy of the 72-bp repeat enhancer is present. We have mapped within the 72-bp repeat the major late promoter component in a 68-bp fragment (located between nucleotides 205 and 272), and found an overlapping 55-bp fragment (located between nucleotides 179 and 234) to have about one-fifth of the late promoter activity. Both the 68- and 55-bp fragments lack some of the core sequence elements required of the 72-bp repeat for transcriptional enhancer activity, and lack the ability to enhance the activity of the SV40 early promoter. The results suggest that the organization of functional units of the 72-bp repeat required for transcriptional enhancement of the early promoter is different from that required for late promoter function. The 21-bp repeat was found to have some late promoter activity located within the origin-distal copy in the absence of the 72-bp repeat. In association with the 21-bp repeat, the otherwise dispensable origin-proximal 22-bp of the 72-bp repeat containing activator protein AP-1 binding site augmented late promoter activity by three- to fourfold.

Antigens, Viral, Tumor↗
PubMed1988

Replication from a proximal simian virus 40 origin is severely inhibited by multiple reiterations of the 72-base-pair repeat enhancer sequence.

In a previous study in our laboratory, the effect of the reiteration frequency of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer on transcription from the proximal SV40 early promoter was investigated (R. Kumar, T. A. Firak, C. T. Schroll, and K. N. Subramanian, Proc. Natl. Acad. Sci. USA 83:3199-3203, 1986). Increasing the enhancer copy number to four increased transcription proportionately; further increments in enhancer copy number reversed this effect, resulting in a decrease in the transcriptional activation. In the present study, the effect of enhancer reiteration on the replication efficiency of plasmids containing the SV40 origin of replication was investigated in transient replication assays in vivo in COS-1 monkey kidney cells producing the SV40 large tumor antigen required for replication. A plasmid containing the SV40 core origin and three copies of the replication-activating, G+C-rich 21-bp repeat promoter element replicated efficiently. Plasmids containing multiple copies of the 72-bp repeat enhancer cloned in head-to-tail linkage adjacent to the 21-bp repeat and the core origin replicated less efficiently; the decrease in replication efficiency could be correlated with the number of copies of the 72-bp repeat; replication was severely curtailed when 10 or more copies of the 72-bp repeat were present. Replication was not significantly inhibited by an increase in the number of copies of the 21-bp repeat to 15 or by the presence of three copies of a 360-bp pBR322 sequence in the immediate vicinity. Multiple copies of the 72-bp enhancer in cis were unable to inhibit replication from a second SV40 origin of replication situated 2 kilobase pairs away from the enhancer reiteration. Replication of four different test plasmids was not inhibited in trans by cotransfection of an excess of a potential competitor plasmid containing a 24-copy reiteration of the 72-bp enhancer. These results indicate that multiple tandem reiterations of the 72-bp enhancer inhibit replication only when they are present in cis adjacent to the origin of replication. Possible explanations for this inhibitory effect, such as an unfavorable local chromatin structure induced by the multimeric enhancer region or reduced or improper communications between factors bound to the multimeric region and the adjacent replication origin, are discussed.

Animals↗
PubMed1988

Direct measurement of CAT activity by incubation of CAT-expressing cells in medium containing chloramphenicol.

We describe a simple, flexible, rapid, sensitive and accurate in vivo assay of the bacterial chloramphenicol acetyltransferase (CAT) enzyme expressed in mammalian cells. The assay is based on the ability of the substrate and products of this enzyme reaction (viz. chloramphenicol and its acetylated derivatives) to equilibrate rapidly between the cells and the surrounding tissue culture medium. We find that chloramphenicol added to the culture medium readily enters the cells and becomes acetylated by the intracellular CAT enzyme. The acetyl derivatives leave the cell and appear rapidly in the culture medium. Due to the large excess of the extracellular compared to the intracellular fluid and due to rapid equilibration of chloramphenicol and its derivatives between them, we find that the bulk of the chloramphenicol and its acetyl derivatives are present in the culture medium at any given time point. Chloramphenicol and its acetylated products are extracted from the medium with ethyl acetate and resolved by thin layer chromatography giving an accurate measurement of the intracellular CAT activity. Sensitive and accurate quantitation of CAT activity in this assay is made possible by the addition of trace amounts of 14C-labeled chloramphenicol to the medium.

Animals↗
PubMed1987

Effects of position and orientation of the 72-base-pair-repeat transcriptional enhancer on replication from the simian virus 40 core origin.

A number of recent studies have reported that in papovaviruses such as simian virus 40 (SV40) and polyomavirus, the replication of the viral DNA in vivo is activated by the viral transcriptional enhancer or promoter sequences. Both viral and cellular transcriptional enhancers are well known for their ability to activate transcription in a position- and orientation-independent manner. In the present study, we investigated the effect of the position and orientation of the SV40 72-base-pair (bp) repeat enhancer on its replication activation function. We constructed plasmids containing one copy each of the SV40 core origin and enhancer placed in either order and orientation and at different distances from each other. We assayed the replication efficiencies of these plasmids in the presence of an internal control plasmid in COS-1 monkey kidney cells producing the SV40 T antigen required for replication. We found that the 72-bp repeat was capable of activating replication equally well in either orientation when placed 8 or 9 bp from the core origin. The activation of replication was totally abolished, and replication efficiencies in most instances were found to be lower than that obtained with the core origin alone, when the 72-bp repeat was separated from the core origin by distances of 99 bp or more. This was in direct contrast to the situation with polyomavirus, in which activation of replication by the homologous enhancer or by the SV40 72-bp repeat enhancer is known to be position independent. We also found that when the SV40 core origin and the 72-bp repeat enhancer were adjacent to each other, efficient activation of replication was obtained only if the end of the core origin containing the 17-bp A + T block was linked with the enhancer. In the other orientation of the core origin, activation of replication was either diminished or abolished. Hypotheses such as alteration of chromatin structure by the enhancer and interaction between trans-acting factors binding to the enhancer and the core origin mediating the activation effect are discussed.

Animals↗
PubMed1986

Activation of gene expression is adversely affected at high multiplicities of linked simian virus 40 enhancer.

We have cloned multiple copies of 72-base-pair (bp) repeat transcriptional enhancer element from simian virus 40 in plasmid vectors upstream from the bacterial chloramphenicol acetyltransferase gene under the control of the simian virus 40 early promoter. Two copies of the 72-bp repeat provided efficient activation of gene expression. Increasing the number of linked 72-bp units to four substantially improved the activation of gene expression, but further addition of enhancers diminished the activation of gene expression proportionally to the number of enhancers added. Enhancer regions containing 10 or more copies of the 72-bp sequence were very inefficient in gene activation. Plasmids containing such expanded enhancer regions also competed less efficiently in vivo for trans-acting enhancer-binding factors. These effects are specific for the enhancer element and are not produced by reiterations of the 21-bp promoter element. Multiple enhancer units placed upstream do not interfere with the accuracy of mRNA initiation directed by the simian virus 40 early promoter in these plasmid constructs but do severely inhibit the initiation of replication at the neighboring simian virus 40 origin of replication that overlaps the early promoter region. These results are consistent with the hypothesis that the structural alterations induced in the DNA by a large number of copies of the enhancer are not favorable for the activation of a linked gene or for the binding of factors believed to mediate the enhancement effect.

Acetyltransferases↗
PubMed1986

Combined high-frequency jet ventilation in a meconium aspiration model.

To evaluate the effectiveness of high-frequency jet ventilation (HFJV) in the treatment of meconium aspiration, 28 puppies were assigned randomly to one of three ventilator techniques. After aspiration of 4 ml/kg of 20% meconium and baseline measurements on conventional ventilation (CV), the animals were ventilated for 6 h with either CV, HFJV, or HFJV combined with slow conventional ventilation (combined HFJV). Arterial and mixed venous blood gases, systemic and pulmonary arterial pressures, pulmonary capillary wedge pressure, and cardiac output were measured. Combined HFJV proved superior to the other two techniques, resulting in significantly better oxygenation and ventilation at a lower mean airway pressure (Paw). Oxygenation with standard HFJV was comparable to CV, producing Paw and PCO2 values intermediate between those of CV and combined HFJV. There were no significant intergroup differences in cardiac output or the incidence of pneumothorax. Improved gas exchange was attributed to alveolar recruitment and prevention of atelectasis. Alteration of the distribution and/or clearance of meconium may also be important.

Animals↗
PubMed1986

Minimal transcriptional enhancer of simian virus 40 is a 74-base-pair sequence that has interacting domains.

We have assayed the ability of segments of the simian virus 40 (SV40) 72-base-pair (bp) repeat enhancer region to activate gene expression under the control of the SV40 early promoter and to compete for trans-acting enhancer-binding factors of limited availability in vivo in monkey CV-1 or human HeLa cells. The bacterial chloramphenicol acetyltransferase and the herpes simplex virus type 1 thymidine kinase genes were used as reporters in these assays. A 94-bp sequence located between SV40 nucleotides 179 and 272, including one copy of the 72-bp repeat, has been termed the minimal enhancer in previous studies. In the present study, we found that the 20-bp origin-proximal region located between nucleotides 179 and 198 was dispensable, since its removal caused only a slight reduction in enhancer activity. However, the deletion of another 4 bp up to nucleotide 202 abolished the enhancer activity. We propose that the minimal enhancer is a 74-bp sequence located between nucleotides 199 and 272, including 52 bp of one copy of the 72-bp repeat and a 22-bp adjacent sequence up to the PvuII site at 272. The nonamer 5'-AAGT/CATGCA-3', which we term the K core, occurred as a tandem duplication around the SphI site at nucleotide 200, and we found that this duplication was essential for enhancement and factor-binding activities. A heterologous core element (which we term the C core), 5'-GTGGA/TA/TA/TG-3', identified earlier (G. Khoury and P. Gruss, Cell 33:313-314, 1983; Weiher et al., Science 219:626-631, 1983) also occurred in duplicate, with one of the copies located within the 22-bp sequence near nucleotide 272 present outside the 72-bp repeat. We provide direct evidence that this 22-bp sequence augments enhancer activity considerably. We also found that in addition to the heterologous interaction occurring normally between the K and C cores within the minimal enhancer, certain homologous interactions were also permitted provided there was proper spacing between the elements.

Animals↗
PubMed1985

Effect of bright light in the hospital nursery on the incidence of retinopathy of prematurity.

The preterm infant is subjected to prolonged exposure to ambient nursery illumination at levels that have been found to produce retinal damage in animals. We prospectively investigated the effect of exposure to light in two intensive care nurseries by comparing the incidence of retinopathy of prematurity among 74 infants from the standard bright nursery environment (median light level, 60 foot-candles [ftc]) with the incidence among 154 infants of similar birth weight for whom the light levels were reduced (median, 25 ftc). There was a higher incidence of retinopathy of prematurity in the group of infants who had been exposed to the brighter nursery lights, particularly in those with birth weights below 1000 g (86 percent vs. 54 per cent, P less than 0.01 by chi-square test). We conclude that the high level of ambient illumination commonly found in the hospital nursery may be one factor contributing to retinopathy of prematurity and that safety standards with regard to current lighting practices should be reassessed.

Birth Weight↗