The helical structure of surfactant peptide KL4 when bound to POPC: POPG lipid vesicles

KL 4 is a 21-residue peptide employed as a functional mimic of lung surfactant protein B, which successfully lowers surface tension in the alveoli. A mechanistic understanding of how KL 4 affects lipid properties has proven elusive as the secondary structure of KL 4 in lipid preparations has not been determined at high resolution. The sequence of KL 4 is based on the C-terminus of SP-B, a naturally occurring helical protein that binds to lipid interfaces. The spacing of the lysine residues in KL 4 precludes the formation of a canonical amphipathic alpha-helix; qualitative measurements using Raman, CD, and FTIR spectroscopies have given conflicting results as to the secondary structure of the peptide as well as its orientation in the lipid environment. Here, we present a structural model of KL 4 bound to lipid bilayers based on solid state NMR data. Double-quantum correlation experiments employing (13)C-enriched peptides were used to quantitatively determine the backbone torsion angles in KL 4 at several positions. These measurements, coupled with CD experiments, verify the helical nature of KL 4 when bound to lipids, with (phi, psi) angles that differ substantially from common values for alpha-helices of (-60, -45). The average torsion angles found for KL 4 bound to POPC:POPG lipid vesicles are (-105, -30); this deviation from ideal alpha-helical structure allows KL 4 to form an amphipathic helix at the lipid interface.     

Investigation of the structural stability of cardiotoxin analogue III from the Taiwan cobra by hydrogen-deuterium exchange kinetics.

The conformational stability of a small ( approximately 7 kDa), all beta-sheet protein, cardiotoxin analogue III (CTX III), from the venom of the Taiwan cobra has been investigated by hydrogen-deuterium (H/D) exchange using two-dimensional NMR spectroscopy. The H/D exchange kinetics of backbone amide protons in CTX III has been monitored at pD 3.6 and 6.6 (at 25 degrees C), for over 5000 h. Examination of H/D exchange kinetics in the protein showed that a number of slowly exchanging residues are in the hydrophobic core of the protein. The average protection factor of the amide protons of residues belonging to the triple-stranded beta-sheet domain is about 20 times greater than that of those in the double-stranded beta-sheet segment. The residues in the C-terminal tail of the molecule, though structureless, have been found to exhibit significant protection against H/D exchange. Comparison of the quenched-flow H/D exchange data on CTX III with those obtained in the present study reveals that the most slowly exchanging portion constitutes the folding core of the protein.     

Molecular genetic analysis of factor X deficiency: gene deletion and germline mosaicism

Summary A case of homozygous factor X deficiency arising from the inheritance of two non-identical gene deletions from heterozygous parents is described. One, a partial gene deletion, was localized to exons VII and VIII by a combination of Southern blotting and polymerase chain reaction (PCR) amplification of exon sequences. The other deletion, of maternal origin, probably involves the entire factor X gene. Restriction fragments associated with the exon VII + VIII deletion were present in three siblings including the homozygous proband. These fragments were however absent from the somatic cells of the father, a finding consistent with germline mosaicism.     

Plasmodium falciparum: genetic polymorphism in apical membrane antigen-1 gene from Indian isolates

A number of stage-specific antigens have been characterized for vaccine development against Plasmodium falciparum malaria. This study presents a comprehensive analysis of the sequence polymorphism in Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) in population samples from the eastern and western parts of India. This is the first study of its kind for the nearly full length PfAMA-1 gene from these regions in India. Our observations confirmed that sequence diversity of PfAMA-1 confines only to point mutations and shows 4-8% variation as compared to the prototypes. As opposed to the previous studies on PfAMA-1, our study revealed a greater degree of polymorphism in the Domain II region of PfAMA-1 protein, though signature for diversifying selection is seen throughout the gene. Our present investigation also indicates a very high degree of variation in the reported T- and B-cell epitopes of PfAMA-1. Few noteworthy and unique observations made in this study are the substitution of Cysteine residues responsible for the disulfide bond structure of the protein and the presence of premature termination after 595 amino acids in 3 of the 13 isolates under consideration. These crucial findings add new perspectives to the future of AMA-1 research and could have major implications in establishing AMA-1 as a vaccine candidate.     

A Simple Computer-aided Device for Monitoring Activity of Small Mammals and Insects

A simple computer-aided device was developed to record the activity of small animals. The device comprises of aktographs, opto-couplers, opto-amplifier units, a data acquisition unit ('Activity Monitor') and a Pentium processor based personal computer (PC) attached to an addition parallel printer port. The aktographs are devices which facilitate organisms to exhibit its natural activity. The aktographs for fruit flies, ants and mice are tubes with food supply and moisture, petri dishes adequately ventilated with arrangements for constant supply of water, sugar or honey solution, and cages attached with running wheels, respectively. Two mutually perpendicular beams of infra-red (IR) emission form a cross-hair detection system across the aktographs and an interruption to these beams creates an electrical signal ('transition') which is amplified using the opto-amplifier unit and sent to the activity monitor which consists of shift registers. The number of moments the IR beams are interrupted is taken as a measure of the amount of activity, based on the assumption that a more active animal would interrupt the IR beams more often compared to a less active one. Programs were written in C++, which facilitated data acquisition and graphical representation in terms of a daily plot of amount of activity as a function of time of the day, and actograms of daily activity were arranged chronologically one below the other. The activity patterns of individual animals (fruit fly, ants, and mice) were monitored using this device. Based on the performance and features of this device, we feel that with some improvement in the software this can substitute some of the existing activity recording devices, which are costly and not flexible.     

A Simple Approach for the Computation of Multiple Periodicities in Biological Time Series

We have described a simple approach for the analysis and isolation of multiple periodicities from a biological time series. For the estimation of the periodicities, we used simulated data and data from ongoing experiments in our laboratory. Two time series were simulated, one which consisted of only white noise and the other consisted white noise along with periodicities of 6, 11, 17 and 23 h, to demonstrate that our method can successfully isolate multiple patterns in a time series. Our method of analysis is objective, simple, flexible and adaptive since it distinctly delineates the individual contribution from an overlap of multiple periodicities. The key features of our method are: (i) identification of a reliable phase reference point, (ii) scanning the time series using a moving window in increments, (iii) use of Siegel's modification of Fisher's method to detect significant periodicit(y)ies in the time series. The use of window sizes of increasing length to examine the time series elegantly reduces noise while identifying periodicities that are otherwise not apparent. Finally, the periodogram can be smoothed in order to normalize the contribution by attendant frequency components within the waveform. A minimum critical value for relative contribution of various frequencies was calculated to delineate the periodicities that contributed significantly to the time series. We executed this method of time series analysis using MS Excel and C.     

Novel N-substituted 4-hydrazino piperidine derivative as a dipeptidyl peptidase IV inhibitor

A novel class of N-substituted 4-hydrazino piperidine derivatives were designed, synthesized and evaluated for DPP IV inhibition. The SAR studies on the N-substituted piperidine led to the discovery of compound 22e as a potent DPP IV inhibitor (IC₅₀ 88 nM), which is highly selective over other peptidases. In vivo efficacy indicates that compound 22e stimulates insulin release in response to glucose load and improves glucose tolerance in n5-STZ and Zucker Diabetic Fatty (ZDF) rats.     

Ubx-Collier signaling cascade maintains blood progenitors in the posterior lobes of the Drosophila larval lymph gland

Drosophila larval hematopoiesis occurs in a specialized multi-lobed organ called the lymph gland. Extensive characterization of the organ has provided mechanistic insights into events related to developmental hematopoiesis. Spanning from the thoracic to the abdominal segment of the larvae, this organ comprises a pair of primary, secondary, and tertiary lobes. Much of our understanding arises from the studies on the primary lobe, while the secondary and tertiary lobes have remained mostly unexplored. Previous studies have inferred that these lobes are composed of progenitors that differentiate during pupation; however, the mechanistic basis of this extended progenitor state remains unclear. This study shows that posterior lobe progenitors are maintained by a local signaling center defined by Ubx and Collier in the tertiary lobe. This Ubx zone in the tertiary lobe shares several markers with the niche of the primary lobe. Ubx domain regulates the homeostasis of the posterior lobe progenitors in normal development and an immune-challenged scenario. Our study establishes the lymph gland as a model to tease out how the progenitors interface with the dual niches within an organ during development and disorders.     

Exploring Missense Mutations in Tyrosine Kinases Implicated with Neurodegeneration

Protein kinases are one of the largest families of evolutionarily related proteins and the third most common protein class of human genome. All the protein kinases share the same structural organization. They are made up of an extracellular domain, transmembrane domain and an intra cellular kinase domain. Missense mutations in these kinases have been studied extensively and correlated with various neurological disorders. Individual mutations in the kinase domain affect the functions of protein. The enhanced or reduced expression of protein leads to hyperactivation or inactivation of the signalling pathways, resulting in neurodegeneration. Here, we present extensive analyses of missense mutations in the tyrosine kinase focussing on the neurodegenerative diseases encompassing structure function relationship. This is envisaged to enhance our understanding about the neurodegeneration and possible therapeutic measures.