A conserved ubiquitin- and ESCRT-dependent pathway internalizes human lysosomal membrane proteins for degradation

The lysosome is an essential organelle to recycle cellular materials and maintain nutrient homeostasis, but the mechanism to down-regulate its membrane proteins is poorly understood. In this study, we performed a cycloheximide (CHX) chase assay to measure the half-lives of approximately 30 human lysosomal membrane proteins (LMPs) and identified RNF152 and LAPTM4A as short-lived membrane proteins. The degradation of both proteins is ubiquitin dependent. RNF152 is a transmembrane E3 ligase that ubiquitinates itself, whereas LAPTM4A uses its carboxyl-terminal PY motifs to recruit NEDD4-1 for ubiquitination. After ubiquitination, they are internalized into the lysosome lumen by the endosomal sorting complexes required for transport (ESCRT) machinery for degradation. Strikingly, when ectopically expressed in budding yeast, human RNF152 is still degraded by the vacuole (yeast lysosome) in an ESCRT-dependent manner. Thus, our study uncovered a conserved mechanism to down-regulate lysosome membrane proteins.

A study of how lysosomal membrane proteins are down-regulated reveals a conserved pathway involving ubiquitination of the membrane protein and subsequent internalization into the lysosome lumen by the ESCRT machinery for degradation.     

Aphidicolin-Induced FRA3B Breakpoints Cluster in Two Distinct Regions

The common fragile site at chromosomal band 3p14.2 (FRA3B) is the most sensitive single site in the human genome to induced chromosomal lesions. This fragile site may predispose chromosome 3p to breakage that is commonly observed in lung, renal, and many other cancers. We previously used aphidicolin induction of FRA3B expression in a chromosome 3-only somatic cell hybrid to generate a series of hybrids with breakpoints in the 3p14.2 region. These breakpoints were localized to two distinct clusters, separated by 200 kb, that lie on either side of a region of frequent breakage within FRA3B as observed by FISH analysis. Seven proximal aphidicolin-induced breakpoints were localized at or near the end of a THE element. The THE-1 element is flanked by LINE andAlurepetitive elements. The eight distal aphidicolin-induced breakpoints clustered in a region capable of forming multiple hairpin-like structures. Thus repetitive elements and hairpin-like structures may be responsible for chromosome fragility in this region.     

Microbial conversion of solasodine to 1-androstene-3,17-dione (AD), a key intermediate for androgen synthesis

The spirosolane side chain of solasodine has been cleaved by cholesterol preinduced Mycobacterium sp. NRRL B-3805 to yield 1-androstene-3,17-dione (AD), a key intermediate for the synthesis of androgenic drugs. Conversion up to 34% has been recorded in shake flask culture after 192 h incubation period using dimethyl-formamide as carrier for solasodine addition.     

Ploidy-dependent genomic stability in the tissue cultures of ornamental Phlox drummondii Hook

Comparisons of the chromosome numbers, 2C nuclear DNA amounts and karyomorphology were made in explant cultures of diploid (2n = 2x = 14) and autotetraploid (2n = 4x = 28) Phlox drummondii. In 6–36 week old calli derived from diploid internodal segment explants, and in cells of root tips regenerated from such callus, marked differences were observed in chromosome number. The chromosome numbers ranged from 2n = 14 to 2n = 100 and DNA amounts from 8.20 to 63.20 pg in the diploid derived callus, while the extent of variation was much reduced in the regenerated roots. In contrast, the autotetraploid cultures were characterised by the maintenance of the same chromosome number and DNA amounts as the mother plant. Modified chromosome structures were not apparent in any of the cultures. The possible reasons for the chromosomal instability at the diploid level and stability at the tetraploid level are discussed.     

DNA fingerprinting in the criminal justice system: an overview

DNA fingerprinting is a powerful technology that has revolutionized forensic science. No two individuals can have an identical DNA pattern except identical twins. Such DNA-based technologies have enormous social implications and can help in the fight against crime. This technology has experienced many changes over time with many advancements occurring. DNA testing is a matter of serious concern as it involves ethical issues. This article describes various trends in DNA fingerprinting and the current technology used in DNA profiling, possible uses and misuses of DNA databanks and ethical issues involved in DNA testing. Limitations and problems prevailing in this field are highlighted.     

Influence of fungal elicitors on production of ajmalicine by cell cultures of Catharanthus roseus

Suspension cultures of Catharanthus roseus (C. roseus) were elicited with fungal cell wall fragments of Aspergillus niger (A. niger), Fusarium moniliforme (F. moniliforme), and Trichoderma viride (T. viride). The effects of elicitor dosage, exposures time, and age of subculture on ajmalicine accumulation were studied. A higher concentration of elicitor extract responded positively to C. roseus suspension cultures. Ajmalicine accumulation increased by about 3-fold when cells were treated with A. niger, F.moniliforme, and T. viride. The maximum ajmalicine production (75 microg g(-1) dry weight (DW)) was observed in cells treated with T. viride. Cell cultures were elicited with 5% preparation of A. niger, F. moniliforme, and T. viride and exposed for 24, 48, 72, and 96 h. for elicitation. Suspension cultures elicited with T. viride for 48 h showed a 3-fold increase (87 microg g(-1) DW) in ajmalicine contents, whereas A. niger and F. moniliforme synthesized a 2-fold increase in alkaloid and yielded 52 and 56 microg g(-1) DW ajmalicine, respectively. C. roseus cells of different age (5,10, 15, 20, and 25 days old) were treated with a 5% elicitor of A. niger, F. moniliforme, and T. viride and investigated elicitors activity at different age of cell cultures. Maximum yield 166 microg g(-1) DW of ajmalicine was synthesized in 20 day old suspension cultures treated with T. viride. A longer period of incubation of cell cultures with elicitors adversely affected the ajmalicine synthesis.     

Phenylpropanoid compounds and disease resistance in transgenic tobacco with altered expression of L-phenylalanine ammonia-lyase

Tobacco plants over-expressing L-phenylalanine ammonia-lyase (PAL(+)) produce high levels of chlorogenic acid (CGA) and exhibit markedly reduced susceptibility to infection with the fungal pathogen Cercospora nicotianae, although their resistance to tobacco mosaic virus (TMV) is unchanged. Levels of the signal molecule salicylic acid (SA) were similar in uninfected PAL(+) and control plants and also following TMV infection. In crosses of PAL(+) tobacco with tobacco harboring the bacterial NahG salicylate hydroxylase gene, progeny harboring both transgenes lost resistance to TMV, indicating that SA is critical for resistance to TMV and that increased production of phenylpropanoid compounds such as CGA cannot substitute for the reduction in SA levels. In contrast, PAL(+)/NahG plants showed strongly reduced susceptibility to Cercospora nicotianae compared to the NahG parent line. These results are consistent with a recent report questioning the role of PAL in SA biosynthesis in Arabidopsis, and highlight the importance of phenylpropanoid compounds such as CGA in plant disease resistance.     

Clofarabine in adult acute leukemias: clinical success and pharmacokinetics

Clofarabine is a deoxyadenosine analog synthesized with the intention of retaining the favorable mechanistic properties of fludarabine and cladribine while eliminating their undesirable characteristics. Phase I studies among 32 patients with acute leukemia defined a maximum tolerated dose (MTD) of 40 mg/m2/d given as a one hour infusion daily for 5 days. The dose limiting toxicity (DLT) was transient hepatotoxicity. In a phase II study, 62 patients with acute leukemias received clofarabine at the MTD over 1 hour daily for 5 days. Twenty patients (32%) achieved complete response (CR), 1 had a partial response (PR), and 9 had a CR but without platelet recovery (CRp), for an overall response rate of 48%. Pharmacokinetic studies in the phase I trial revealed marked heterogeneity in peak levels of clofarabine among patients at the end of infusion, however; there was a linear, dose dependent increase in clofarabine concentration in the plasma. Pharmacodynamically, at the MTD, DNA synthesis was inhibited by more than 80% at the end of infusion. In phase II studies, the relationship between the pharmacokinetics of clofarabine triphosphate accumulation and clinical response at the MTD was explored, revealing an accumulation advantage of the cytotoxic triphosphate in leukemia cells of responders. The circulating leukemia blasts of patients who respond to clofarabine therapy exhibited a favorable pharmacokinetic profile. In conclusion, clofarabine is an active agent in the treatment of acute leukemias and MDS, and cellular pharmacokinetics has prognostic significance.