Profile of organ dysfunction and predictors of mortality in severe scrub typhus infection requiring intensive care admission.

BACKGROUND AND AIMS: Scrub typhus, a zoonotic rickettsial infection, is an important reason for intensive care unit (ICU) admission in the Indian subcontinent. We describe the clinical profile, organ dysfunction, and predictors of mortality of severe scrub typhus infection. MATERIALS AND METHODS: Retrospective study of patients admitted with scrub typhus infection to a tertiary care university affiliated teaching hospital in India during a 21-month period. RESULTS: The cohort (n = 116) aged 40.0 ± 15.2 years (mean ± SD), presented 8.5 ± 4.4 days after symptom onset. Common symptoms included fever (100%), breathlessness (68.5%), and altered mental status (25.5%). Forty-seven (41.6%) patients had an eschar. Admission APACHE-II score was 19.6 ± 8.2. Ninety-one (85.2%) patients had dysfunction of 3 or more organ systems. Respiratory (96.6%) and hematological (86.2%) dysfunction were frequent. Mechanical ventilation was required in 102 (87.9%) patients, of whom 14 (12.1%) were solely managed with non-invasive ventilation. Thirteen patients (11.2%) required dialysis. Duration of hospital stay was 10.7 ± 9.7 days. Actual hospital mortality (24.1%) was less than predicted APACHE-II mortality (36%; 95% Confidence interval 32-41). APACHE-II score and duration of fever were independently associated with mortality on logistic regression analysis. CONCLUSIONS: In this cohort of severe scrub typhus infection with multi-organ dysfunction, survival was good despite high severity of illness scores. APACHE-II score and duration of fever independently predicted mortality.

Rat sperm immobilisation effects of a protein from Ricinus communis (Linn.): an in vitro comparative study with nonoxynol-9.

Previous study conducted in our department showed that 50% ethanolic extract of the root of Ricinus communis possess reversible antifertility effect and a 62-kDa protein (Rp) from this extract is responsible for the antifertility effects. In this study, we compared the spermicidal effect of this Rp with nonoxynol-9 (N-9) in vitro. The sperm immobilisation studies showed that 100 µg ml(-1) of Rp was able to immobilise the sperms completely within 30 s. Sperm revival test revealed that the spermicidal effect was irreversible. There was also a significant reduction in sperm viability and hypo-osmotic swelling in Rp and N-9 treated groups in comparison with the control. In Rp and N-9 treated groups, the number of acrosome-reacted cells was found to be high and also caused agglutination of the spermatozoa, indicating the loss of intactness of the plasma membrane, which was further supported by the significant reduction in the activity of membrane bound 5'-nucleotidase, acrosomal acrosin. In short, the protein Rp possesses spermicidal activity in vitro and its effects are similar to that of nonoxynol 9.

Spermicidal action of a protein isolated from ethanolic root extracts of Achyranthes aspera: an in vitro study.

A previous study conducted in our department, showed that 50% ethanolic extract of the roots of Achyranthes aspera possess spermatotoxic effects. Preliminary studies also revealed that the active principle may be a protein. In this study a 58 kDa Achyranthes protein (Ap) was isolated from Achyranthes aspera using standard protocols and their effects on the rat sperm was studied in vitro in comparison with nonoxynol-9 (N-9). The sperm immobilization studies showed that about 150 µg of Ap was able to immobilize sperms completely within seconds at a lower concentration than N-9 (250 µg). The sperm revival test revealed that the spermicidal effect was irreversible. There was also a significant reduction in sperm viability and hypo-osmotic swelling in the Ap-treated and N-9 treated groups in comparison to the control. In the Ap and N-9 treated groups the number of acrosome reacted cells were found to be high and it also caused agglutination of the sperms indicating the loss of intactness of the plasma membrane which was further supported by the significant reduction in the activity of membrane bound 5' nucleotidase and acrosin enzyme. Hence this study showed that the protein isolated from the roots of Achyranthes aspera possess spermicidal activity in vitro and can act as a spermicide similar to that of nonoxynol 9. Ap also possessed spermicidal activity against human sperms in vitro.

Effects on spermatogenesis in swiss mice of a protein isolated from the roots of Ricinus communis (Linn.) (Euphorbiaceae).

This study was aimed to evaluate the effect on spermatogenesis of a 62 kDa protein (Rp) isolated from 50% ethanolic extract of the root of Ricinus communis in mice. A dose response study in mice revealed that 25mg/kg body weight/day was the most effective dose. Swiss strain mature male mice of 30 days old were divided into two group namely control and Rp treated (25mg/kg body weight/day). The study showed that sperm motility and count were decreased significantly in the treated group as compared to the control. The fertility index of the treated groups was reduced by 100%. The activity of HMG Co A reductase and cholesterol were increased significantly in the treated group. The testicular activities of 3ßHSD, 17ßHSD, glucose 6-phosphate dehydrogenase and malic enzyme and the level of serum testosterone were decreased significantly in the treated group. The expression of 3ßHSD and 17ßHSD were decreased and the expression of StAR increased significantly in the treated group as compared to the control. Proteolytic digestion of the native protein with trypsin and chymotrypsin showed that the proteolytic cleavage did not affect the spermicidal action of Rp. Hence this study can be concluded that Rp impaired spermatogenesis in vivo by suppressing the production of testosterone.

Role of cardiac isoform of alpha-2 macroglobulin in diabetic myocardium.

Earlier studies from one of the investigator's laboratory have demonstrated the presence of a high molecular weight protein (182 kDa) in the blood serum of laboratory animals subjected to pressure-induced cardiac hypertrophy and suggested that this protein may be involved in the development of cardiac hypertrophy. Studies have shown that this protein is also involved in earlier stages of cardiac complications associated with diabetes, but the role of this protein in diabetic heart is less understood. So we aimed to check whether this protein is having any protective role in diabetic heart. The protein was purified from serum of rats induced with cardiac hypertrophy and the purified protein was injected through tail vein of diabetic rats for further studies. The results of various antioxidant enzymes and the TBARS levels have indicated the antioxidant activity of this protein. Real-time PCR analysis of gene expression revealed the upregulation of certain muscle-specific genes like ß-MHC, MLC-2, and skeletal α actin in diabetic group and also in presence of 182-kDa protein. The results further showed a down regulation of genes such as cardiac α-actin and α- MHC implicating the role of this protein in the development of cardiac hypertrophy in diabetes. Increased cardiac hypertrophy as revealed by the expression of various genes and improved antioxidant potential in presence of 182 kDa protein in diabetes at the earlier stages is beneficial for counteracting the myocardial damage associated with diabetes.

Photodynamic effects of two hydroxyanthraquinones.

The aim of this work was to investigate the photodynamic action of electron-rich anthraquinones, viz., cynodontin (CYN) and cynodontin-5,8-dimethylether (CYNM). Both optical and EPR methods are used to detect the generation of singlet oxygen. Based on RNO bleaching, relative to rose bengal (RB), singlet oxygen generating efficiencies of CYN and CYNM are derived to be 0.055 and 0.254, respectively. The formation of superoxide anion via electron transfer to O2 was monitored by optical spectroscopy, using SOD-inhibitable cytochrome c reduction assay. The production of O2-* is enhanced in the presence of electron donors such as EDTA and NADH. Photolysis of CYN and CYNM in DMSO, in the presence of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), generates a multi-line EPR spectrum, characteristic of spin adduct mixture of O2-* and *OH. Both optical and ESR measurements indicate that O2-* (Type I) and 1O2 (Type II) paths are involved in CYN and CYNM photodynamic action.

Toxicity of PCB 1232 on mitochondria of fish Arius caelatus (Valenciennes).

Mitochondrial proteins and phospholipids were estimated and SDH, Na(+)-K(+)-ATPase and Mg(2+)-ATPase activities were analysed in the gill, liver and heart tissues of PCB 1232 (sublethal doses) treated fish A. caelatus. Protein and phospholipids were found to be decreased significantly and SDH, Na(+)-K(+)-ATPase, Mg(2+)-ATPase and other enzyme systems displayed an inverse relationship with PCB dosage. Statistical analysis was carried out to indicate the relationship between sublethal doses of varying concentration and the activities of the enzyme systems involved in energy metabolism. The studies indicated impairment in mitochondrial functions.

Cardiac isoform of alpha-2 macroglobin, a novel serum protein, may induce cardiac hypertrophy in rats.

Earlier studies from this laboratory have identified a novel high molecular weight (182 kDa) serum protein suggested to be involved in the development of cardiac hypertrophy. In the present case the role of this novel serum protein in the development of pressure-induced cardiac hypertrophy and the molecular events associated with it in experimental rats has been investigated. Multiple injections of this purified protein intravenously (through tail vein) into the normal animals lead to the development of cardiac hypertrophy and this is accompanied by an induction of muscle specific genes such as that of MLC2 and beta-MHC characteristic of pressure overloaded heart. Further, the hypertrophy-specific serum protein has been found to be identical to rat alpha-2 macroglobulin (alpha-2M) in molecular weight (182 kDa) and in its appearance in blood serum. alpha-2M is an acute phase serum protein that increases markedly after inflammatory stimuli in hepatocytes in liver and gets secreted into the blood. The studies at present suggest that the 182kDa serum protein that appeared during the early stage of development of cardiac hypertrophy in aorta constricted rats is a glycoprotein localized in the heart that showed immunological cross reactivity with alpha-2M and is expressed in the heart as evinced by Northern blot analysis. Further this protein showed certain differences from rat alpha-2M under denaturing conditions in isoelectric focusing and partial peptide mapping. Partial peptide sequencing of the internal peptides of tryptic digest of 182 kDa showed 100% identity of the sequences with alpha-2M sequences. Rat alpha-2M does not, however, have any influence on the development of cardiac hypertrophy and its antibody does not cross react with the 182 kDa protein. These data suggest that the 182 kDa protein that may play an indispensable role in the development of cardiac hypertrophy in experimental rats is cardiac specific, and may be an isoform of liver alpha-2M belonging to macroglobulin family.

A novel serum protein of molecular weight 182 kDa: a molecular marker for an early detection of increased left ventricular mass in patients with cardiac hypertrophy.

BACKGROUND: Earlier studies in our laboratory have shown the presence of a cardiac-hypertrophy-specific high-molecular-weight protein of 182 kDa in the sera of laboratory rats which were subjected to aortic stenosis. On the basis of a number of criteria, these studies have pointed out that this protein may be a molecular signal of hypertrophic growth in the aorta-constricted animals. Further, a similar high-molecular-weight protein has been observed in the sera of normal humans and patients with cardiac anomalies. We have tried to correlate the levels of 182 kDa serum protein with various parameters such as age, severity of hypertrophy and left ventricular muscle (LVM) mass in patients with cardiac hypertrophy. METHODS: Two hundred and ten patients with left and right ventricular hypertrophy evidenced by clinical history, physical examination, electrocardiogram and echocardiogram were selected for the study. Sandwich enzyme-linked immunosorbent assay (ELISA) technique was used to quantify the 182 kDa serum protein in the sera of patients by using anti-rat 182 kDa protein antibodies. RESULTS: The 182 kDa protein levels in the serum correlated with the age and stage of the LVM mass of hypertrophied heart in patients. It was noted that this protein was significantly elevated in early and moderate stages of cardiac hypertrophy and decreased when the hypertrophy became severe in patients only up to 40 years of age, whereas no significant difference exists in 182 kDa protein levels between normal individuals and patients with cardiac hypertrophy aged over 40 years. CONCLUSION: The level of this protein could be an early molecular marker identifying the stages of increase in LVM mass in patients with cardiac hypertrophy, below 40 years of age. The induction of 182 kDa protein levels in human serum may be an age-dependent phenomenon.

Epidemiology of gastro-intestinal nematodes of sheep in wet tropical conditions in Malaysia.

A study on the seasonal variations in the population structure of Haemonchus contortus and Trichostronglyus colubriformis was conducted for a period of 12 months in a typical large scale sheep farm on improved pasture in Peninsular Malaysia which has a wet tropical climate. Successive groups of helminth-free tracer lambs were grazed for 4 weeks together with naturally infected sheep and were necropised for worm counts 2 weeks after their removal from the pasture. The monthly populations of H. contortus fluctuated slightly except in May and August during which more worms were found in the tracer animals. The numbers of T. colubriformis were comparatively high from October to December 1992 and again in March 1993, low during April and June 1992. Small numbers of hypobiotic larvae of H. contortus were detected in the tracer animals. Development and survival of infective larvae of H. contortus and T. colubriformis on pasture were investigated by spreading faeces containing eggs on grass plots in October 1993, February and May 1994. Development of the eggs to the infective larvae occurred within one week and their survival times were 7 weeks in the 3 experiments. The potential for control by rotational grazing is discussed.

Expression of proto-oncogenes, genes for muscle specific isoforms and heat shock protein (HSP) -70 gene in hypertrophied cardiac muscles from patients with atrial septal defect or tetralogy of Fallot.

Studies on the expression of proto-oncogenes and muscle specific genes in hypertrophied human hearts have shown that proto-oncogenes such as c-myc, c-fos and c-ras are activated in both atrial and ventricular tissues of patients with atrial septal defect (ASD) and tetralogy of Fallot (TOF). Although the expression of muscle specific genes such as MLC2 and skeletal alpha-actin are induced the expression of tissue specific cardiac actin remained the same in both the control and diseased tissues. Further, an increased synthesis of messengers of heat shock protein gene-HSP70 was observed in the ventricular tissues of TOF patients, with out much of a change in atrial tissues of patients with ASD.

Serum protein of 135-kDa molecular weight--a molecular signal for cardiac hypertrophy.

A serum protein, similar to the 135-kDa protein which appears during experimentally induced cardiac hypertrophy in rats, was identified in human serum by Western blot analysis using anti-rat 135-kDa protein antibody. The rat protein antibody gave a very strong positive signal when reacted with sera obtained from cardiac patients, suggesting an induced level of this protein in patients' sera when compared to normal sera from healthy individuals. Multiple injections of polyclonal anti-rat 135-kDa protein antibody to aorta-constricted animals completely inhibited the development of cardiac hypertrophy, suggesting that this protein could be hypertrophy specific. This was further substantiated by the decrease in the expression of a molecular marker such as the beta myosin heavy chain gene in aorta-constricted but antibody-injected animals. Further, the presence of high levels of this protein in first- and third-day neonatal animals is also suggestive of the hypertrophy-specific nature of this protein as the heart is subjected to high pressure overload immediately after birth.

Effect of cytosol on the regulation of expression of myosin heavy chain genes during cardiac hypertrophy.

In vitro translation of RNA transported from the rat heart nuclei has suggested that the transport of translatable messages from hypertrophic heart nuclei is greater than from sham-operated heart nuclei. An increased translation activity was observed in cell-free system with RNA transported from sham-operated heart nuclei in presence of hypertrophic heart cytosol, than from sham-operated heart cytosol. Similar results were obtained when myosin heavy chain (MHC) mRNA in the transported RNA was analyzed by slot-blot hybridization using beta cDNA probe. Immunoprecipitation analysis of the translated products using beta MHC isozyme specific antibody indicated that the increased levels of beta MHC specific mRNA in the RNA transported from sham-operated heart nuclei in presence of hypertrophic heart cytosol than sham-operated heart cytosol. Direct quantitation of alpha and beta MHC messengers by slot-blot hybridization analysis of transported RNA using oligomeric probes corresponding to the 3' untranslated regions of alpha and beta MHC mRNAs revealed that an increased transport of both alpha and beta MHC specific mRNAs from sham-operated heart nuclei in the presence of hypertrophic heart cytosol occurs, of which beta MHC mRNA is more than that of alpha MHC. In contrast, slot-blot hybridization analysis of the radioactive RNA synthesized during transcription in vitro in nuclei obtained from sham-operated as well as hypertrophic hearts has shown an increased synthesis of alpha in sham nuclei and that of beta in hypertrophic heart nuclei. These results suggest that both transcriptional and post-transcriptional regulation may be operative in the expression of alpha and beta MHC genes during the development of cardiac hypertrophy.

Purification and characterization of a high-molecular-weight protein induced in rat serum during the development of cardiac hypertrophy.

A relatively high-molecular-weight polypeptide was found in rat serum within 6 h after aortic constriction in experimental animals. This polypeptide persists for about 7 days of the postoperative period and disappears at later stage of hypertrophy (40%). Further, fractionation and purification of this protein through DEAE-Sepharose and gel filtration chromatography have revealed that this protein is a single polypeptide and its relative molecular weight is 135 kDa. Immunoprecipitation and immunofluorescence microscopic analysis have indicated the presence of the above polypeptide in the nuclear fraction of heart cells. Studies on phosphorylation in vitro have revealed that this protein is a phosphoprotein. DNase I sensitivity and hybridization using a muscle specific gene probe have indicated the involvement of this protein in template associated changes in heart nuclei. Further the possibility of this protein being synthesized by heart cells indicates that this protein could traverse back and forth between heart cells and the extracellular fluid, suggesting an autocrine/paracrine role for this protein during the development of cardiac hypertrophy.

Role of cytosol in the stimulation of RNA transport in vitro during cardiac hypertrophy in rats.

The 100,000 g supernatant isolated from hypertrophic hearts on fractionation by (NH4)2SO4 and DEAE-cellulose chromatography showed an enhanced RNA-transport activity when incubated with isolated nuclei from sham-operated hearts in vitro. Proteins of Mr 73,000, 68,000, 43,000 and 32,000 are enriched in the DEAE-cellulose fractions exhibiting maximal transport activity, and they are phosphorylatable. Pretreatment of the cytosol with antibodies to the Mr-68,000 and -32,000 proteins decreases the transport activity of the cytosol from 14% to 4.25%. Proteins of Mr 73,000, 68,000, 43,000 and 32,000 are translocated from the cytosol to the nuclear envelope under conditions of RNA transport in vitro. Our results here suggest that at least two of these proteins, those of Mr 68,000 and 32,000, play an indispensible role in the nucleocytoplasmic RNA transport in vitro. By making use of a specific myosin heavy-chain B-gene probe and hybridization, we have also shown the effect of cytosol on the transport of myosin heavy-chain mRNA from nucleus to cytosol.

Changes associated with rat heart chromatin during cardiac hypertrophy.

Studies with N-Pyrenemaleimide, a probe which binds to H3 among histones and tryptophan residues of nonhistone proteins and subsequently emits fluorescence, showed that during developing cardiac hypertrophy, conformational changes due to DNA-histone interactions may be maximum at an early stage of hypertrophy (17% hypertrophy) while the changes due to non-histone protein(s) interactions may be maximum at a latter stage (28% hypertrophy). In vitro transcription analysis in isolated nuclei obtained from normal and hypertrophic hearts showed that nuclei obtained from 40% hypertrophic hearts had a maximum incorporation ability than nuclei obtained from 17% or 28% hypertrophic hearts. Analysis of histones and 0.35 M NaCl extractable nuclear proteins by single dimensional polyacrylamide gel electrophoresis did not reveal any changes in these proteins when obtained from normal and different stages of hypertrophic hearts. DNase I sensitivity studies with nuclei obtained from normal and hypertrophic hearts and with reconstituted nuclei, showed that changes in 0.35 M NaCl extractable proteins may contribute to the respective DNase I sensitivity of various hypertrophic nuclei. These studies also indicated that though nuclei obtained from 40% hypertrophic hearts showed maximum incorporation during in vitro transcription, the DNase I sensitivity is maximum only in nuclei obtained from 28% hypertrophic hearts and not from 40% hypertrophic hearts.