Testis-specific ATP synthase peripheral stalk subunits required for tissue-specific mitochondrial morphogenesis in <Emphasis Type="Italic">Drosophila</Emphasis>

Background

In Drosophila early post-meiotic spermatids, mitochondria undergo dramatic shaping into the Nebenkern, a spherical body with complex internal structure that contains two interwrapped giant mitochondrial derivatives. The purpose of this study was to elucidate genetic and molecular mechanisms underlying the shaping of this structure.

Results

The knotted onions (knon) gene encodes an unconventionally large testis-specific paralog of ATP synthase subunit d and is required for internal structure of the Nebenkern as well as its subsequent disassembly and elongation. Knon localizes to spermatid mitochondria and, when exogenously expressed in flight muscle, alters the ratio of ATP synthase complex dimers to monomers. By RNAi knockdown we uncovered mitochondrial shaping roles for other testis-expressed ATP synthase subunits.

Conclusions

We demonstrate the first known instance of a tissue-specific ATP synthase subunit affecting tissue-specific mitochondrial morphogenesis. Since ATP synthase dimerization is known to affect the degree of inner mitochondrial membrane curvature in other systems, the effect of Knon and other testis-specific paralogs of ATP synthase subunits may be to mediate differential membrane curvature within the Nebenkern.

     

Tuneable luminescence properties of EDTA‐assisted ZnS:Mn nanocrystals from a yellow‐orange to a red emission band

Luminescence technology has been improved with the help of semiconductor nanoparticles that possess novel optical and electrical properties compared with their bulk counterpart. The aim of this study was to design semiconductor nanocrystals in their pure (ZnS) or doped form (ZnS:Mn) with different concentrations of Mn²⁺ ions by a wet chemical route stabilized by ethylenediamine tetra‐acetic acid (EDTA) and to evaluate their luminescence properties. The nanocrystals were characterized by physicochemical techniques such as X‐ray diffraction (XRD), High‐resolution transmission electron microscopy (HRTEM), selected area electron diffraction (SEAD), EDS, and ultraviolet (UV)–visible light and photoluminescence (PL) studies. These results showed the presence of cubic phase and spherically shaped nanocrystals. A blue shift with respect to their bulk counterpart was observed. PL emission spectra were observed with a fixed blue peak and the yellow‐orange bands were red shifted towards the red region under the same excitation wavelength. The orange‐red bands were attributed to the radiation transition of electrons in 3d5 unfilled shells of Mn²⁺ ions [⁴T₁(⁴G)‐⁶A₁(⁶S)]; the ZnS matrix varied with Mn²⁺ concentration. Shift and increase in the intensity of the PL and absorption bands were observed with increase in Mn content. The study showed that Mn²⁺‐doped ZnS nanocrystal emission bands can be tuned from the yellow‐orange to the red regions under a controlled synthesis process and could be used as promising luminescent emitters in the biology field upon functionalization with suitable materials. Further studies on construction with various other materials will be useful for practical applications.     

A three-step strategy for targeting drug carriers to human ovarian carcinoma cells in vitro.

To improve tumor-to-tissue ratios of anticancer agents in radioimmunotherapy, a three-step targeting approach was used to deliver biotinylated liposomes to human ovarian cancer cells (NIH:OVCAR-3, SK-OV-3) in vitro. Targeting was based upon the use of two antibodies specific for the CA-125 antigen that is highly expressed on NIH:OVCAR-3 cells but not expressed on SK-OV-3 cells. Briefly, the approach consists of prelabeling target cells with biotinylated anti-CA-125 antibody and FITC-labeled streptavidin (SAv) prior to administration of biotinylated liposomes containing a marker dye for visualization by confocal laser scanning microscopy (CLSM). In addition, the two anti-CA-125 antibodies (B27.1 and B43.13) were labeled with FITC and incubated with ovarian cancer cells at 37 degrees C from 30 min to 24 h to study binding and uptake kinetics. Shedding kinetics of bound antibody from tumor cells was performed using radiolabeled B27.1. Results demonstrated that both B27.1 and B43.13 specifically bound to the cell surface of OVCAR-3 cells but not to SK-OV-3 cells. Biotinylation, FITC-labeling and radiolabeling of the antibodies did not compromise immunoreactivity. Less than 6% of the bound B27.1 was shed from tumor cells by 4 h following incubation, and the antibody-antigen complex resided predominantly on the cell surface by 4 h at 37 degrees C with slow internalization by 12-24 h. Biotinylated, conventional liposomes were specifically and effectively delivered to OVCAR-3 cells prelabeled with biotinylated B27.1 and SAv. The slow internalization and shedding properties of these antibodies are useful for multistep pretargeting methods. Thus, a modified targeting strategy, utilizing a bispecific antibody and liposomes, may be feasible for radioimmunoliposomal therapy of ovarian cancer.     

Synthesis of peptides employing 9-fluorenylmethyl chloroformate as a coupling agent

Summary The synthesis of peptides employing 9-fluorenylmethyl chloroformate (Fmoc-Cl) as a coupling agent has been described. The method is simple, efficient and rapid. All the peptides have been obtained in good yield (70–95%). Furthermore, both the¹H NMR and the HPLC studies on Fmoc-Phg-Phe-OMe and Fmoc-D-Phg-Phe-OMe revealed that the coupling is free from racemization.     

Density‐dependence in common tree species in a tropical dry forest in Mudumalai,southern India

Abstract. Density‐dependence in tree population dynamics has seldom been examined in dry tropical forests. Using long‐term data from a large permanent plot, this study examined 16 common species in a dry tropical forest in southern India for density‐dependence. Employing quadrat‐based analyses, correlations of mortality, recruitment and population change with tree densities were examined. Mortality in 1–10 cm diameter trees was largely negatively correlated with conspecific density, whereas mortality in > 10 cm diameter trees was positively correlated. Mortality was, however, largely unaffected by the basal area and abundance of heterospecific trees. Recruitment was poor in most species, but in Lagerstroemia microcarpa (Lythraceae), Tectona grandis (Verbenaceae) and Cassia fistula (Fabaceae), species that recruited well, strong negative correlations of recruitment with conspecific basal area and abundance were found. In a few other species that could be tested, recruitment was again negatively correlated with conspecific density. In Lagerstroemia, recruitment was positively correlated with the basal area and abundance of heterospecific trees, but these correlations were non‐significant in other species. Similarly, although the rates of population change were negatively correlated with conspecific density they were positive when dry‐season ground fires occurred in the plot. Thus, the observed positive density‐dependence in large‐tree mortality and the negative density‐dependence in recruitment in many species were such that could potentially regulate tree populations. However, repeated fires influenced density‐dependence in the rates of population change in a way that could promote a few common species in the tree community.     

Transposon-Like Organization of the Plasmid-Borne Organophosphate Degradation (opd) Gene Cluster Found in Flavobacterium sp.

Several bacterial strains that can use organophosphate pesticides as a source of carbon have been isolated from soil samples collected from diverse geographical regions. All these organisms synthesize an enzyme called parathion hydrolase, and in each case the enzyme is encoded by a gene (opd) located on a large indigenous plasmid. These plasmids show considerable genetic diversity, but the region containing the opd gene is highly conserved. Two opd plasmids, pPDL2 from Flavobacterium sp. and pCMS1 from Pseudomonas diminuta, are well characterized, and in each of them a region of about 5.1 kb containing the opd gene shows an identical restriction pattern. We now report the complete sequence of the conserved region of plasmid pPDL2. The opd gene is flanked upstream by an insertion sequence, ISFlsp1, that is a member of the IS21 family, and downstream by a Tn3-like element encoding a transposase and a resolvase. Adjacent to opd but transcribed in the opposite direction is an open reading frame (orf243) with the potential to encode an aromatic hydrolase somewhat similar to Pseudomonas putida TodF. We have shown that orf243 encodes a polypeptide of 27 kDa, which plays a role in the degradation of p-nitrophenol and is likely to act in concert with opd in the degradation of parathion. The linkage of opd and orf243, the organization of the genes flanking opd, and the wide geographical distribution of these genes suggest that this DNA sequence may constitute a complex catabolic transposon.     

The Mechanism of Action of Multidrug-Resistance-Linked P-Glycoprotein

P-glycoprotein (Pgp), the ATP-binding cassette (ABC) transporter, confers multidrug resistance to cancer cells by extruding cytotoxic natural product amphipathic drugs using the energy of ATP hydrolysis. Our studies are directed toward understanding the mechanism of action of Pgp and recent work deals with the assessment of interaction between substrate and ATP sites and elucidation of the catalytic cycle of ATP hydrolysis. The kinetic analyses of ATP hydrolysis by reconstituted purified Pgp suggest that ADP release is the rate-limiting step in the catalytic cycle and the substrates exert their effect by modulating ADP release. In addition, we provide evidence for two distinct roles for ATP hydrolysis in a single turnover of Pgp, one in the transport of drug and the other in effecting conformational changes so as to reset the transporter for the next catalytic cycle. Detailed kinetic measurements determined that both nucleotide-binding domains behave symmetrically and during individual hydrolysis events the ATP sites are recruited in a random manner. Furthermore, only one nucleotide site hydrolyzes ATP at any given time, causing (in this site) a conformational change that drastically decreases (>30-fold) the affinity of the second site for ATP-binding. Thus, the blocking of ATP-binding to the second site while the first one is in catalytic conformation appears to be the basis for the alternate catalytic cycle of ATP hydrolysis by Pgp, and this may be applicable as well to other ABC transporters linked with the development of multidrug resistance.     

Characterisation of a starch-hydrolysing enzyme of Aspergillus niger

A UV-induced mutant strain of Aspergillus niger (CFTRI-1105-U9) overproduced a starch-hydrolysing enzyme with properties characteristically different from the known amylases of the fungus. The purified enzyme of 4.0 pI had an apparent molecular mass of 125 kDa and it dextrinised starch and then saccharified the dextrins. Patterns of the enzyme activity on starch, resulting in glucose at 60 °C and glucose, maltose and maltodextrins at 70 °C as primary products, suggested significant applications for the enzyme in starch-processing industries. Received: 29 October 1998 / Received revision: 11 January 1999 / Accepted: 19 January 1999     

Dosimetric comparison of normal breathing and deep inspiration breath hold technique for synchronous bilateral breast cancer using 6MV flattened beam and flattening filter free beam

BackgroundThe present study was to investigate the usefulness of deep inspiration breath hold (DIBH) in bilateral breast patients using 6MV flattened beam (FB) and flattening filter free beam (FFFB).Materials and methodsTwenty bilateral breast cancer patients were simulated, using left breast patients treated with DIBH technique. CT scans were performed in the normal breathing (NB) and DIBH method. Three-dimensional conformal radiotherapy (3DCRT) and volumetric arc therapy (VMAT) plans were generated.ResultsIn our study the best organ at risk (OAR) sparing is achieved in the 3DCRT DIBH plan with adequate PTV coverage (V₉₅ ≥ 47.5 Gy) as compared to 6MV FB and FFFB VMAT DIBH plans. The DIBH scan plan reduces the heart mean dose significantly at the rate of 49% in 3DCRT (p = 0.00) and 22% in VMAT (p = 0.010). Similarly, the DIBH scan plan produces lesser common lung mean dose of 18% in 3DCRT (p = 0.011) and 8% in VMAT (0.007) as compared to the NB scan. The conformity index is much better in VMAT FB (1.04 ± 0.04 vs. 1.04 ± 0.05), p =1.00 and VMAT FFFB (1.04 ± 0.05 vs. 1 ± 0.24, p = 0.345) plans as compared to 3DCRT (1.63 ± 0.2 vs. 1.47 ± 0.28, p = 0.002). The homogeneity index of all the plans is less than 0.15. The global dmax is more in VMAT FFFB DIBH plan (113.7%). The maximum MU noted in the NB scan plan (478 vs. 477MU, 1366 vs. 1299 MU and 1853 vs. 1788 MU for 3DCRT, VMAT FB and VMAT FFFB technique as compared to DIBH scan.ConclusionWe recommend that the use of DIBH techniques for bilateral breast cancer patients significantly reduces the radiation doses to OARs in both 3DCRT and VMAT plans.