Molecular genetics of cell death in the nematode Caenorhabditis elegans

In C. elegans, cell death can be readily studied at the cellular, genetic, and molecular levels. Two types of death have been characterized in this nematode: (1) programmed cell death, which occurs as a normal component in development; and (2) pathological cell death which occurs aberrantly as a consequence of mutation. Analysis of mutations that disrupt programmed cell death in various ways has defined a genetic pathway for programmed cell death which includes genes that perform such functions as the determination of which cells die, the execution of cell death, the engulfment of cell corpses, and the digestion of DNA from dead cells. Molecular analysis is providing insightinto the nature of the molecules that function in these aspects of programmed cell death. Characterization of some genes that mutate to induce abnormal cell death has defined a novel gene family called degenerins that encode putative membrane proteins. Dominant alleles of at least two degenerin genes, mec-4 and deg-1, can cause cellular swelling and late onset neurodegeneration of specific groups of cells. © 1992 John Wiley & Sons, Inc.     

Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.     

Impaired Surfactant Production by Alveolar Epithelial Cells in a SCID-hu Lung Mouse Model of Congenital Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) is the leading viral cause of birth defects and life-threatening lung-associated diseases in premature infants and immunocompromised children. Although the fetal lung is a major target organ of the virus, HCMV lung pathogenesis has remained unexplored, possibly as a result of extreme host range restriction. To overcome this hurdle, we generated a SCID-hu lung mouse model that closely recapitulates the discrete stages of human lung development in utero. Human fetal lung tissue was implanted into severe combined immunodeficient (CB17-scid) mice and inoculated by direct injection with the VR1814 clinical isolate of HCMV. Virus replication in the fetal lung was assessed by the quantification of infectious virus titers and HCMV genome copies and the detection of HCMV proteins by immunohistochemistry and Western blotting. We show that HCMV efficiently replicated in the lung implants during a 2-week period, forming large viral lesions. The virus productively infected alveolar epithelial and mesenchymal cells, imitating congenital infection of the fetal lung. HCMV replication triggered apoptosis near and within the viral lesions and impaired the production of surfactant proteins in the alveolar epithelium. Our findings highlight that congenital and neonatal HCMV infection can adversely impact lung development, leading to pneumonia and acute lung injury. We have successfully developed a small-animal model that closely recapitulates fetal and neonatal lung development and provides a valuable, biologically relevant tool for an understanding of the lung pathogenesis of HCMV as well as other human respiratory viruses. Additionally, this model would greatly facilitate the development and testing of new antiviral therapies for HCMV along with select human pulmonary pathogens.     

Collagen targeting using multivalent protein-functionalized dendrimers

Collagen is an attractive marker for tissue remodeling in a variety of common disease processes. Here we report the preparation of protein dendrimers as multivalent collagen targeting ligands by native chemical ligation of the collagen binding protein CNA35 to cysteine-functionalized dendritic divalent (AB₂) and tetravalent (AB₄) wedges. The binding of these multivalent protein constructs was studied on collagen-immobilized chip surfaces as well as to native collagen in rat intestinal tissues. To understand the importance of target density we also created collagen-mimicking surfaces by immobilizing synthetic collagen triple helical peptides at various densities on a chip surface. Multivalent display of a weak-binding variant (CNA35-Y175K) resulted in a large increase in collagen affinity, effectively restoring the collagen imaging capacities for the AB₄ system. In addition, dissociation of these multivalent CNA35 dendrimers from collagen surfaces was found to be strongly attenuated.     

Occurrence of Horizontal Gene Transfer of PIB-type ATPase Genes among Bacteria Isolated from the Uranium Rich Deposit of Domiasiat in North East India

Uranium (U) tolerant aerobic heterotrophs were isolated from the subsurface soils of one of the pre-mined U-rich deposits at Domiasiat located in the north-eastern part of India. On screening of genomic DNA from 62 isolates exhibiting superior U and heavy metal tolerance, 32 isolates were found to be positive for P_IB-type ATPase genes. Phylogenetic incongruence and anomalous DNA base compositions revealed the acquisition of P_IB-type ATPase genes by six isolates through horizontal gene transfer (HGT). Three of these instances of HGT appeared to have occurred at inter-phylum level and the other three instances indicated to have taken place at intra-phylum level. This study provides an insight into one of the possible survival strategies that bacteria might employ to adapt to environments rich in uranium and heavy metals.     

Protoplast formation and regeneration in <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis>

Method for production and regeneration of Lactobacillus delbrueckii protoplasts are described. The protoplasts were obtained by treatment with a mixture of lysozyme and mutanolysin in protoplast buffer at pH 6.5 with different osmotic stabilizers. The protoplasts were regenerated on deMan, Rogosa and Sharpe (MRS) with various osmotic stabilizers. Maximum protoplast formation was obtained in protoplast buffer with sucrose as an osmotic stabilizer using a combination of lysozyme (1 mg/ml) and mutanolysin (10 μg/ml). Maximum protoplast regeneration was obtained on MRS medium with sucrose (0.5 M) as an osmotic stabilizer. The regeneration medium was also applicable to other species of lactobacilli as well. This is, to our knowledge, the first report on protoplast formation and efficient regeneration in case of L. delbrueckii.     

Cluster analysis of behavioural and event-related potentials during a contingent negative variation paradigm in remitting-relapsing and benign forms of multiple sclerosis

Background  

Event-related potentials (ERPs) may be used as a highly sensitive way of detecting subtle degrees of cognitive dysfunction. On the other hand, impairment of cognitive skills is increasingly recognised as a hallmark of patients suffering from multiple sclerosis (MS). We sought to determine the psychophysiological pattern of information processing among MS patients with the relapsing-remitting form of the disease and low physical disability considered as two subtypes: 'typical relapsing-remitting' (RRMS) and 'benign MS' (BMS). Furthermore, we subjected our data to a cluster analysis to determine whether MS patients and healthy controls could be differentiated in terms of their psychophysiological profile.     

Role of Melanin in Release of Extracellular Enzymes and Selection of Aggressive Isolates of Bipolaris sorokiniana in Barley

Eighteen barley isolates of Bipolaris sorokiniana belonging to wild and clonal type of black, mixed and white subpopulations were quantitatively assayed for their melanin content and aggressiveness with respect to production of some of the extracellular enzymes such as cellulase, pectinase, amylase and protease. Cellulase and pectinase constituted major portion of the enzymes recovered from the black, mixed and white isolates. Enzyme production and aggressiveness were relatively higher in melanin devoid or low melanin isolates. The melanin deficient isolates were also differentiated from black and mixed isolates on the basis of variation in internal transcribed spacer region of the ribosomal DNA. Higher enzyme productions positively correlated with area under disease progress curve (AUDPC) and lesion development. Melanin content was negatively correlated with extracellular enzymes and aggressiveness of the isolates. Based on melanin content, lesion size, AUDPC and extracellular enzymes, the isolates were grouped in two major clusters (I and II) with further division of cluster II into two sub-clusters (II-A and II-B). The results appears to indicate a possible role of melanin in release of extracellular enzymes and hence in evolution and selection of aggressive isolates of B. sorokiniana in barley.     

Folate binding and transport by rat kidney brush-border membrane vesicles

[3H]Pteroylglutamic acid (PteGlu) uptake was studied using brush-border membrane vesicles isolated from rat kidney. Results on the uptake of [3H]PteGlu by brush-border membrane vesicles incubated in media of increasing osmolarities demonstrated that uptake was contributed by two components, intravesicular transport and membrane binding. Both the components of the uptake exhibited similar pH dependence, with maxima at pH 5.6, and were found to be saturable mechanisms with Km values of 6.7.10(-7) and 11.2.10(-7) M, respectively. These studies show that PteGlu is transported by isolated rat kidney brush-border membrane vesicles in a manner consistent with a saturable system and that a binding component may be functionally associated with this.