Homodimerization of human bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) and its functional implications

Genetic lesions of bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) completely or partially abolish hepatic bilirubin glucuronidation, causing Crigler-Najjar syndrome type 1 or 2, respectively. Clinical observations indicate that some mutant forms of human UGT1A1 (hUGT1A1) may be dominant-negative, suggesting their interaction with the wild-type enzyme. To evaluate intermolecular interaction of hUGT1A1, Gunn rat fibroblasts were stably transduced with hUGT1A1 cDNA. Gel permeation chromatography of solubilized microsomes suggested dimerization of hUGT1A1 in solution. Nearest-neighbor cross-linking analysis indicated that, within microsomal membranes, hUGT1A1 dimerized more efficiently at pH 7.4 than at pH 9. Two-hybrid analysis in yeast and mammalian systems demonstrated positive interaction of hUGT1A1 with itself, but not with another UGT isoform, human UGT1A6, which differs only in the N-terminal domain. Dimerization was abolished by deletion of the membrane-embedded helix from the N-terminal domain of hUGT1A1, but not by substitution of several individual amino acid residues or partial deletion of the C-terminal domain. A C127Y substitution abolished UGT1A1 activity, but not its dimerization. Coexpression of mutagenized and wild-type hUGT1A1 in COS-7 cells showed that the mutant form markedly suppressed the catalytic activity of wild-type hUGT1A1. Homodimerization of hUGT1A1 may explain the dominant-negative effect of some mutant forms of the enzyme.     

Healthcare infrastructure, contraceptive use and infant mortality in Uttar Pradesh, India

This paper analyzes data on approximately 30,000 women from a survey in Uttar Pradesh in 1995 together with the data from surveys of public and private providers of healthcare and family planning services. A framework was developed for analyzing the effects of quality of services on utilization, and for understanding the gradual evolution of the healthcare infrastructure. The empirical results from logistic regressions for use of female sterilization and IUD showed significant effects of quality of services in government and private hospitals, and of socioeconomic variables such as education, caste, and an index of household possessions. Secondly, models for infant mortality of children born in the preceding 3-year period showed significant effects of socioeconomic variables, quality of healthcare services and birth spacing. Lastly, analysis of data at a more aggregated (Primary Sampling Unit) level indicated differential effects of economic development on the quality of services available in the public and private facilities.     

Differential response of NaCl-Resistant mutants of the cyanobacterium Nostoc muscorum to salinity and osmotic stresses

Summary In the parent strain of Nostoc muscorum, the percentage survival, nitrogenase activity and oxygenic photosynthesis were severely impaired by NaCl (ionic) and sucrose (non-ionic) stresses. Spontaneously occurring NaCl-Resistant mutant clones of the cyanobacterium N. muscorum were found to exhibit differential responses under ionic and non-ionic stresses. One of the mutants (NaCl-R) was found to show resistance in terms of percentage survival, nitrogenase activity and oxygenic photosynthesis under saline (ionic) as well as osmotic (non-ionic) stresses and showing compatible solute strategy for such adaptation. Another mutant (Na⁺-R) was found to show resistance only to salinity stress and showed an enhanced Na⁺-efflux system driven by H⁺. The Na⁺-R mutant differed from the NaCl-R mutant strain in the sense that it was sucrose sensitive.     

pPAC-ResQ: A yeast-bacterial shuttle vector for capturing inserts from P1 and PAC clones by recombinogenic targeted cloning

We have developed a method to capture inserts from P1 and P1 artificial chromosome (PAC) clones into a yeast-bacteria shuttle vector by using recombinogenic targeting. We have engineered a vector, pPAC-ResQ, a derivative of pClasper, which was previously used to capture inserts from yeast artificial chromosome clones. pPAC-ResQ contains DNA fragments flanking the inserts in P1 and PAC vectors as recombinogenic ends. When linearized pPAC-ResQ vector and P1 or PAC DNA are cotransformed into yeast, recombination between the two leads to the transfer of inserts into pPAC-ResQ. pPAC-ResQ clones thus obtained can be further modified in yeast for functional analysis and shuttled to Escherichia coli to produce large quantities of cloned DNA. This approach provides a rapid method to modify P1/PAC clones for functional analysis.     

In vitro anti- biofilm and anti-bacterial activity of Sesbania grandiflora extract against Staphylococcus aureus

The main objective of this research is to investigate the anti-biofilm and anti-bacterial activity of Sesbania grandiflora (S. grandiflora) against Staphylococcus aureus. S. grandiflora extract were prepared and analyzed with UV –Vis spectroscopy, Fourier transform infrared spectroscopy, Dynamic light scattering. Biofilm forming pathogens were identified by congo-red assay. Quantification of Extracellular polymeric substance (EPS) particularly protein and carbohydrate were calculated. The efficacy of the herbal extract S. grandiflora and its inhibition against the pathogenic strain of S. aureus was also evaluated. The gradual decrease or disappearance of peaks reveals the reduction of protein and carbohydrate content in the EPS of S. aureus when treated with S. grandiflora. The antibacterial activity of S. grandiflora extract against the bacterial strain S. aureus showed that the extract were more active against the strain. To conclude, anti-biofilm and antibacterial efficacy of S. grandiflora plays a vital role over biofilm producing pathogens and act as a good source for controlling the microbial population.     

Technical note: comparison of the Maresh reference data with the who international standard for normal growth in healthy children

The Maresh reference data on stature and long bone lengths in a sample of healthy middle-class children from Denver, Colorado [Maresh: Am J Dis Child 66 (1943) 227-257; Maresh: Am J Dis Child 89 (1955) 725-742; Maresh: Human growth and development (1970) p 155-200], have been used extensively by biological anthropologists to estimate juvenile age and body size using skeletal elements and to assess growth in skeletal series from different ethnic populations or archaeological cultural groups. How well these data reflect the potentially diverse growth patterns of healthy human populations from different geographic areas is unknown. Similarly, the efficacy of using the Maresh reference data to estimate stunting prevalence in prehistoric populations is unknown. This report presents the results from a comparison of the Maresh data on supine length and standing height to the World Health Organization (WHO) international child growth standard. The WHO growth standard is meant to depict typical human growth under optimal conditions and can be used to assess children worldwide, regardless of ethnicity and socioeconomic status. The results from this comparison indicate that although the Maresh reference data generally conform to the WHO standard, reflecting a normal human growth pattern, and therefore serve as a suitable reference for comparative studies of growth patterns, these reference data are not suitable for estimating stunting prevalence.     

A potent and specific immunotoxin for tumor cells expressing disialoganglioside GD2

Summary Monoclonal antibody 14G2a (anti-GD₂) reacts with cell lines and tumor tissues of neuroectodermal origin that express disialoganglioside GD₂. mAb 14G2a was coupled to the ribosome-inactivating plant toxin gelonin with the heterobifunctional cross-linking reagentN-succinimidyl-3(2-pyridyldithio)propionate. The activity of the immunotoxin was assessed by a cell-free translation assay that confirmed the presence of active gelonin coupled to 14G2a. Data from an enzyme-linked immunosorbent assay demonstrated the specificity and immunoreactivity of the 14G2a-gelonin immunotoxin, which was identical to that of native 14G2a. Assays for complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) revealed that these functional properties of the native 14G2a antibody were also preserved in the 14G2a-gelonin immunotoxin. The gelonin-14G2a immunotoxin was directly cytotoxic to human melanoma (A375-M and AAB-527) cells and was 1000-fold more active than native gelonin in inhibiting the growth of human melanoma cells in vitro. The augmentation of tumor cell killing of 14G2a-gelonin immunotoxin was examined with several lysosomotropic compounds. Chloroquine and monensin, when combined with 14G2a-gelonin immunotoxin, augmented its cytotoxicity more than 10-fold. Biological response modifiers such as tumor necrosis factor and interferon and chemotherapeutic agents such as cisplatinum andN,N-bis(2-chloroethyl)-N-nitrosourea (carmustine) augmented the cytotoxicity of 14G2a-gelonin 4- to 5-fold. The results of these studies suggest that 14G2a-gelonin may operate directly by both cytotoxic efforts and indirectly by mediating both ADCC and CDC activity against tumor cells; thus it may prove useful in the future for therapy of human neuroectodermal tumors.Research conducted, in part, by the Clayton Foundation for Research     

Expression and characterization of isoform 1 of human mitochondrial elongation factor G

Elongation factor G (EF-G) catalyzes the translocation step of protein biosynthesis. Genomic analysis suggests that two isoforms of this protein occur in mitochondria. The region of the cDNA coding for the mature sequence of isoform 1 of human mitochondrial EF-G (EF-G1(mt)) has been cloned and expressed in Escherichia coli. The recombinant protein has been purified to near homogeneity by chromatography on Ni-NTA resins and cation exchange high performance liquid chromatography. EF-G1(mt) is active on both bacterial and mitochondrial ribosomes. Human EF-G1(mt) is considerably more resistant to fusidic acid than many bacterial translocases. A molecular model for EF-G1(mt) has been created and analyzed in the context of its relationship to the translocases from other systems.