Splenic infarct in falciparum malaria.
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Child↗
Biomedical subjects
S Agarwal
Publications and source records attributed to S Agarwal.
cDNA and genomic cloning and expression of the P48 monocytic differentiation/activation factor, a Mycoplasma fermentans gene product.
P48 is a 48 kDa monocytic differentiation/activation factor previously purified from the conditioned medium of the Reh human pre-B cell leukaemia cell line. It induces growth arrest and differentiation of HL-60 human promyelocytic leukaemia cells along the monocytic pathway and the production of the cytokines interleukin 1, tumour necrosis factor-alpha and interleukin 6 in human monocytes and monocytic cell lines. The cDNA for P48 was cloned from Reh cellular RNA using 3' reverse amplification of cDNA ends. Southern blot probing with P48 cDNA revealed hybridization with DNA from Reh and Molt-4 cells, but not with DNA from human peripheral blood mononuclear cells. Subsequent studies using PCR and Southern analysis revealed P48 sequences in DNA isolated from Mycoplasma fermentans but not M. hominis, M.iowae, M.synoviae or M.lypophilum. Although initial studies using Mycoplasma culture and hybridization techniques had failed to reveal Mycoplasma infection in our Reh and Molt-4 cell lines, subsequent PCR studies using Mycoplasma genus-specific rRNA primrs revealed Mycoplasma sequences in these cell lines. Using the P48 cDNA probe, we isolated a genomic clone from M. fermentans DNA which was found to be 98.5% identical with the P48 cDNA clone, and the deduced amino acid sequence agreed with N-terminal microsequencing data for P48 protein purified from the Reh cell line conditioned medium. The 5' end of the gene has a number of consensus sequences characteristic of prokaryotic genes, and the deduced amino acid sequence has a number of features suggesting that P48 is a lipoprotein. The P48 cDNA was expressed in pMAL in Escherichia coli, and the 60 kDa expressed fusion protein was found to react with anti-P48 antibodies on Western blots. This is consistent with a pMAL fusion protein representing the sum of the 42 kDa maltose-binding protein and 18 kDa of P48 recombinant protein, suggesting that native P48 has significant post-translational modification. Consistent with this, Northern blot studies revealed a single 1 kb transcript. The recombinant fusion protein was found to possess anti-proliferative activity against HL-60 cells, and antibodies against recombinant P48 were found to block the biological activity of native P48 isolated from conditioned medium. These studies demonstrate that P48, a molecule with immunomodulatory and haematopoietic differentiation activities, is derived from M. fermentans or a closely related species. P48 may be important in the pathophysiology of Mycoplasma infections and may be useful in dissecting the mechanisms involved in mammalian haematopoietic cell differentiation, immune function and cytokine biosynthesis.
Amino Acid Sequence↗
Non-conservative mutations are well tolerated in the globular region of yeast histone H4.
Yeast histone H4 has been mutagenized at several positions which participate in the globular core of the nucleosome. The native protein contains residues at those positions which are invariant or highly conserved over all known H4 sequences, whether from yeast, Tetrahymena or higher eukaryotes. Nonetheless the protein is tolerant of non-conservative mutations. At the level of cell function the mutant proteins cause no significant change in length of the cell cycle of mating efficiency. At the level of chromatin structure no effect is observed on the internucleosomal spacing of chromatin or the pattern of hydroxyl radical cleavage of nucleosomal DNA.
Amino Acid Sequence↗
Products of ozonized arachidonic acid potentiate the formation of DNA single strand breaks in cultured human lung cells.
In this study we examined the potential for environmental levels of ozone (03) to degrade arachidonic acid (AA), a polyunsaturated fatty acid abundantly present in the lung, into products that can produce DNA single strand breaks (ssb) in cultured human lung cells. Human lung fibroblasts were incubated with 60 microM AA that had been previously exposed to and degraded by 0.4 ppm 03 (1 hr.) Incubation of the cells with 03-exposed AA (but not with vehicle alone) for 1 hr at 4 degrees C and 37 degrees C produced 555 and 245 rad-equivalents of DNA ssb, respectively, as determined by the DNA alkaline elution technique. These breaks were completely eliminated when the ozonized AA solution was incubated with catalase prior to cell treatment, indicating that h202 was solely responsible for damaging DNA. Superoxide dismutase bovine serum albumin, or heat-inactivated catalase showed little, if any, inhibitory activity. The H202 content of the ozonized AA (31 +/- 4 microM) could account for only about 40% of the observed breaks. Potentiation of the H202-induced DNA ssb persisted after removal of the carbonyl substances by chromatographic procedures, suggesting that the non-carbonyl component of ozonized AA was the responsible component for inducing augmentation of the observed increases in DNA ssb. Ozonized AA also induced DNA ssb in cultures of the human bronchial epithelial cell line BEAS-2B. Again, these breaks were shown to exceed levels that could be attributed to the presence of H202 alone. These results indicate that products of ozonized AA can interact to potentiate DNA ssb in human lung cells.
Animals↗
Fetal radiation exposure is minimal after pelvimetry by modified digital radiography.
OBJECTIVE: Our purpose was to determine the maximal fetal exposure to radiation by use of thermoluminescent dosimeters when pelvic measurements were performed by standard or modified digital radiography. STUDY DESIGN: Digital radiography of the pelvis was performed according to a standard technique. Lithium fluoride thermoluminescent dosimeters were positioned on the patient's skin to quantitate the maximal amount of radiation exposure to the fetus. The standard technique often included a portion of fetal vertex. The axial view technique was modified to use an angle of inclination of 17 to 29 degrees relative to the vertical axis. RESULTS: Digital radiography was well tolerated and interpretable images were consistently obtained. The maximal dose to the fetal vertex by use of the standard digital axial slice was 465 mrad. By modifying the standard digital technique and using an angle of inclination of 17 to 29 degrees it was possible to obtain an axial section without including any portion of the fetal vertex. This reduced the maximal total dose to the fetal vertex to 55 mrad, which is less than the background radiation exposure to the fetus over a 9-month period from natural sources. CONCLUSION: Data reported indicate that total fetal radiation exposure is minimal after pelvimetry by digital radiography. Incorporating the modification of the angle for the axial slice, as reported here, resulted in a further significant decrease in fetal radiation exposure.
Female↗
HLA sharing, anti-paternal cytotoxic antibodies and MLR blocking factors in women with recurrent spontaneous abortion.
OBJECTIVE: To study the prevalence of HLA sharing between spouses and its correlation with presence of antipaternal cytotoxic antibody (APCA) and mixed lymphocyte reaction (MLR) blocking factors in recurrent spontaneous aborters (RSA). DESIGN: Study was carried out at Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGI), Lucknow, from 1988-1992. Hundred couples with 3 or more consecutive recurrent spontaneous abortions and equal number of age, parity and ethinically matched normal controls were selected for studying HLA, APCA and MLR blocking factors. Meta-analysis was performed using standard formula and significance was tested by Chi-square analysis. RESULTS: Significant HLA sharing was observed in couples with RSA at A and DR loci compared to normal controls (p < 0.001). Twenty-seven point eight percent of couples with RSA were positive for APCA compared to 49% of controls (p < 0.01). MLR blocking factors were detected in 26% couples with RSA compared to 78% in controls (p < 0.001). An inverse correlation between HLA sharing and APCA and MLR positivity was demonstrated. CONCLUSION: The study supports that greater HLA sharing between spouses, associated with lack of an appropriate immune response to them could be responsible for RSA.
Abortion, Habitual↗
Abdominal pain in a young girl.
A 14-year-old girl was admitted to our hospital with peri-umbilical pain associated with obstipation and vomiting. Plain erect X-rays of the abdomen revealed multiple air fluid levels. A supine X-ray is shown in this figure.
Abdominal Pain↗
Renal failure with skeletal abnormalities.
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Adult↗
Tuberculosis--an underestimated cause of ileal perforation.
One hundred and thirteen specimens of Ileum received over the last 3 years (1990-92) were reviewed histologically and analysed to determine the aetiology of perforation. Perforations were seen in all age groups varying from one month to 75 years with a peak incidence during 3rd and 4th decades. Males were more frequently affected. Perforations were single or multiple involving the whole ileum. The highest incidence (52) was seen in enteric fever. Next in frequency (19) was tuberculosis. In 23 cases no specific aetiology could be identified. Other causes of perforation were injury and obstruction resulting from diverticulosis, intussusception adhesions and worms. A significantly high incidence (16.8%) of tuberculous perforation is noteworthy.
Adolescent↗
Detection and analysis of an alternatively spliced isoform of interleukin-6 mRNA in peripheral blood mononuclear cells.
Oligonucleotide primers for human interleukin-6 (IL-6) that bracketed the entire coding region of the gene were used in reverse transciptase-polymerase chain reaction (RT-PCR) studies to examine lL-6 expression in peripheral blood mononuclear cells (PBMC). In addition to the predicted 0.64-kb RT-PCR product, a second 0.45-kb product was observed. Cloning and dideoxy sequence analysis of this product revealed evidence for an alternatively spliced lL-6 transcript lacking exon II. Further RT-PCR analysis using forward primers ending at or one base before the exon I donor splice site again yielded both products. Additional primers were designed and successfully used to selectively distinguish the two forms of IL-6 transcript. Both transcripts were prominent in peripheral blood monocytes and lymphocytes, whereas only the 0.64-kb, full-length transcript was prominent in the lL-6-producing 5637 (human bladder carcinoma) cell line. Northern analysis revealed, in addition to the predominant 1.3-kb transcript, several minor transcripts at 1.9 to 4.8 kb that hybridized with the alternatively spliced cDNA probe but not with an exon II probe. Western analysis revealed lL-6 polypeptides of predicted size (26 to 29 kD) in culture medium from PBMC, while showing an immunoreactive band at 17 kD in cell lysates. These findings suggest the existence of an alternatively spliced form of lL-6 mRNA, which would encode for a polypeptide missing the gp130 interactive (signal-transducing) domain contained in exon II while retaining the lL-6 receptor (p80) domain. Such a molecule could in theory function as a natural antagonist of lL-6, as it would be expected to bind to the IL-6 receptor but not lead to signal transduction.
Amino Acid Sequence↗
Differential oxidative damage to mitochondrial proteins during aging.
The level of mitochondrial protein oxidative damage, indicated by the protein carbonyl content, has been previously shown to increase with age. The objective of this study was to determine whether such age-associated damage is random or whether some proteins are relatively more susceptible. Mitochondrial proteins were analyzed by gel filtration. High molecular weight proteins were found to be relatively more oxidatively damaged during aging. Results are interpreted to indicate that protein oxidative damage during aging is not random. The question of the validity of the HPLC method for the measurement of protein carbonyls is discussed in light of a recent report questioning its reliability.
Aging↗
Reconstitution of signal transduction from the membrane to the nucleus in a baculovirus expression system: activation of Raf-1 leads to hypermodification of c-jun and c-fos via multiple pathways.
We have attempted to dissect signaling pathways involved in transmitting activating signals from the cell surface to the nucleus by reconstituting them in the baculovirus/Sf9 cell system. We have used this system to coexpress different combinations of the critical signaling proteins pp60v-src, p21v-ras, Raf-1 and ERK-1 and assayed the effects of resulting signaling cascades on the modifications of coexpressed transcription factors c-jun or c-fos. We observe that activation of ERK-1 via Raf-1 and p21ras dependent signals can result in the hyperphosphorylation of c-jun. In contrast, c-fos appears to be the target of two Raf-1 activated modifying signals: one independent of ERK-1 and the other dependent on ERK-1 stimulation. Thus, coexpression of c-fos with pp60v-src, p21v-ras or constitutively active forms of Raf-1 results in a dramatic reduction of its electrophoretic mobility in the absence of coexpressed ERK-1. Activation of this ERK-1-independent pathway together with the ERK-1 dependent pathway that modifies c-jun results in additional modification of c-fos. Our observation of a Raf-1 activated, ERK-independent signaling pathway is consistent with previous reports that constitutively active Raf-1 can, in some cell types, result in transformation or differentiation without activation of ERKs. Our data indicate the presence of multiple Raf-1 activated pathways that lead to modification of transcription factors.
3T3 Cells↗
Oxidative stress and aging in the Mongolian gerbil (Meriones unguiculatus).
The objective of this study was to determine if aging in the gerbil, Meriones unguiculatus, is associated with elevation in the level of oxidative stress. Studies were conducted on the brain, heart, kidney, liver and testis of young (3-6 months), adult (15 months), and old (23-25 months) animals. Oxidative damage to proteins, measured as the concentration of protein carbonyls and loss of activity of glucose-6-phosphate dehydrogenase, and to DNA, measured as the concentration of 8-hydroxydeoxyguanosine, increased with age of the animals. There was no appreciable age-related change in the activity of alkaline proteases, which preferentially degrade oxidized protein. Rates of mitochondrial superoxide anion radical and hydrogen peroxide generation also increased with age, most notably in the heart. Antioxidative defenses, measured as activities of superoxide dismutase, catalase and glutathione peroxidase and concentration of glutathione, did not exhibit a uniform pattern of age-related changes. However, when the antioxidative potential of the tissue homogenates was measured as their susceptibility to undergo protein oxidation, in response to experimentally-induced oxidative stress, using X-irradiation, tissues of the old animals were significantly more vulnerable than those of the young animals. Results of this study are interpreted to indicate: (i) that the level of molecular oxidative damage to DNA and proteins increases with age, and (ii) that the increased oxidative damage is due to both an an elevation in the rates of oxidant generation and an increase in the susceptibility of tissues to oxidative damage.
Aging↗
Simultaneous overexpression of copper- and zinc-containing superoxide dismutase and catalase retards age-related oxidative damage and increases metabolic potential in Drosophila melanogaster.
The objective of this study was to elucidate the nature of the mechanisms by which overexpression of copper- and zinc-containing superoxide dismutase (Cu,Zn-SOD) and catalase extends life span of Drosophila melanogaster. Experimental flies containing single extra doses of Drosophila Cu,Zn-SOD and catalase genes were compared with appropriate controls at different ages. Overexpression of Cu,Zn-SOD and catalase caused a retardation in the accumulation of 8-hydroxydeoxyguanosine during aging and in response to the exposure of live flies to x-rays. The age-related loss of glucose-6-phosphate dehydrogenase activity (Glu-6-P dehydrogenase) and the increase in the rate of mitochondrial H2O2 generation were less steep in the experimental than control flies. The rate of in vivo oxygen consumption in the latter two-thirds of life span was higher in the experimental than in the control flies. Furthermore, the metabolic potential, or the total amount of oxygen consumed during an average life span, was about 30% greater in the experimental than the control flies. Altogether, results of this study indicate that overexpression of Cu,Zn-SOD and catalase ameliorates the age-related accumulation of molecular oxidative damage and susceptibility to such damage in response to acute oxidative stress while prolonging the metabolic life of flies. The concept that oxidative stress is a causal factor in the aging process is supported.
8-Hydroxy-2'-Deoxyguanosine↗
Effects of peroxidative stress and age on the concentration of a deoxyguanosine-malondialdehyde adduct in rat DNA.
The effect of age and peroxidative stress on the concentration of a deoxyguanosine malondialdehyde adduct (dG-MDA) in rat tissues was investigated. Vitamin E deficiency had no effect on the dG-MDA content of liver DNA in rats fed a diet containing 10% corn oil. When 2% cod liver oil was added to this diet, the dG-MDA content of liver DNA doubled in the positive controls fed a high level of vitamin E (100 ppm dl-alpha-tocopherol), and there was a further increase when vitamin E was deleted. Neither iron nitrilotriacetate administration nor choline deficiency had any effect on the dG-MDA content of liver DNA. Carbon tetrachloride had a lowering effect. The failure of iron or carbon tetrachloride administration and of vitamin E deficiency to increase liver dG-MDA is consistent with their failure in previous experiments to affect the urinary excretion of dG-MDA. In contrast, these forms of peroxidative stress produce large increments in the urinary excretion of MDA adducts with lysine, reflecting increased formation and degradation of MDA-modified proteins. DNA appears to be protected from modification by MDA produced at extranuclear sites. The frequency of dG-MDA in different tissues of 4-month-old rats varied markedly: brain >> liver > kidneys and testes. Higher concentrations of dG-MDA were found in the liver and kidneys, but not the testes, of 25-month-old rats. The determinants of the concentration of dG-MDA in DNA merit further investigation.
Aging↗
Synthesis of proinflammatory cytokines by human gingival fibroblasts in response to lipopolysaccharides and interleukin-1 beta.
We have examined the ability of gingival fibroblasts (GF) to participate in inflammatory response and function as accessory immune cells. The accessory immune function of GF cells was evaluated by their ability to elaborate proinflammatory cytokines following stimulation with lipopolysaccharides and interleukin-1 beta (IL-1 beta). Using three separate clonally derived and characterized human gingival fibroblast (GF) cell lines, we demonstrate that LPS from Actinobacillus actinomycetemcomitans (Aa) and Escherichia coli (Ec) induce mRNA and synthesis of proinflammatory cytokines, IL-1 beta, IL-6 and IL-8. IL-1 beta activation of GF cells showed that IL-1 beta non only induces the expression of IL-6, IL-8 and TNF-alpha, but also acts in an autocrine manner of GF cells and induces IL-1 beta expression. Furthermore, the continuous presence of IL-1 beta in GF cell cultures did not down regulate the response of GF cells to IL-1 beta. Pretreatment of GF cells with IL-1 beta resulted in the enhanced synthesis of TNF-alpha in response to additional IL-1 beta. These findings indicate that GF cells, in addition to providing structural support, may also function as accessory immune cells and play an important role in the initial inflammatory reaction as well as in the amplification of immune response.
Aggregatibacter actinomycetemcomitans↗
Differential expression of IL-1 beta, TNF-alpha, IL-6, and IL-8 in human monocytes in response to lipopolysaccharides from different microbes.
Macrophages respond to bacterial lipopolysaccharides (LPS) and activate several host defense functions through production of mediators. However, it is not clear whether the degree of macrophage responsiveness to different sources of LPS is equivalent to or varies with the source of LPS. Therefore, in this report, we examined the extent of the human monocyte response to LPS derived from two oral pathogens, Actinobacillus actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg). Additionally, due to its well-established ability to activate monocytes, we used LPS from Escherichia coli (Ec). Human monocytes, when activated with a specific source of LPS, exhibited rapid expression of mRNA for IL-1 beta, TNF-alpha, and IL-8, which was followed by IL-6, as measured by RNA-PCR. Moreover, the expression of mRNA for these cytokines was followed by cytokine synthesis. Monocytes from the same subject, when activated with LPS from Pg, Aa, or Ec expressed quantitatively different levels of mRNA and proteins for all four cytokines. A given LPS induced either high or low expression of the battery of cytokines tested, indicating that the expression of these pro-inflammatory cytokines may be regulated by a single or a cluster of gene(s). However, no apparent differences in the time course of mRNA expression for these cytokines were observed in response to any of the LPS tested. Furthermore, the relative ability of the different sources of LPS to induce mRNA for cytokines varied throughout a wide range of LPS concentrations. This suggests that differences exist in the sensitivity of monocytes to a specific LPS, rather than in the kinetics of the secretory process itself.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
LPS responsiveness in periodontal ligament cells is regulated by tumor necrosis factor-alpha.
Gingival fibroblasts function as accessory immune cells and are capable of synthesizing cytokines in response to lipopolysaccharides (LPS) from Gram-negative microbes. Recently, we have isolated, cloned, and characterized two cell lines which exhibit characteristics of periodontal ligament (PDL) cells. In this report, we demonstrate that PDL cells showing osteoblast-like phenotype are not LPS-responsive cells. However, treatment of PDL cells with tumor necrosis factor-alpha (TNF-alpha) inhibits the expression of their osteoblast-like characteristics. As a consequence of this TNF-alpha-induced phenotypic change, PDL cells become LPS-responsive, i.e., synthesize several pro-inflammatory cytokines in response to LPS. These phenotypic changes occur at concentrations of TNF-alpha that are frequently observed in tissue exudates during periodontal inflammation, suggesting a physiological significance for these in vitro observations. It is of interest that TNF-alpha-induced phenotypic changes in PDL cells are transient, since removal of rhTNF-alpha from the supernatants of PDL cell cultures results in re-acquisition of the osteoblast-like characteristics and lack of LPS responsiveness of PDL cells. These results suggest that TNF-alpha, by regulating the PDL cell functions, may allow these cells to participate in the disease process as accessory immune cells at the expense of their structural properties.
Alkaline Phosphatase↗