Recent advancements in the development of anti-tuberculosis drugs

Modern chemotherapy has significantly improved patient outcomes against drug-sensitive tuberculosis. However, the rapid emergence of drug-resistant tuberculosis, together with the bacterium’s ability to persist and remain latent present a major public health challenge. To overcome this problem, research into novel anti-tuberculosis targets and drug candidates is thus of paramount importance. This review article provides an overview of tuberculosis highlighting the recent advances and tools that are employed in the field of anti-tuberculosis drug discovery. The predominant focus is on anti-tuberculosis agents that are currently in the pipeline, i.e. clinical trials.     

Molecular characterisation and expression of two venom allergen-like protein genes in Heterodera glycines.

Secretory proteins encoded by genes expressed in the oesophageal gland cells of plant-parasitic nematodes have key roles in nematode parasitism of plants. Two venom allergen-like protein cDNAs (designated hg-vap-1 and hg-vap-2)were isolated from Heterodera glycines gland cell cDNA libraries. Both cDNAs hybridised to genomic DNA of H. glycines in Southern blots. The hg-vap-1 cDNA contained an open reading frame encoding 215 amino acids with the first 25 amino acids being a putative secretion signal. The hg-vap-2 cDNA contained an open reading frame encoding 212 amino acids with the first 19 amino acids being a putative secretion signal. Genes of hg-vap-1 and hg-vap-2 contained four introns, which ranged in size from 44 to 574 bp, and five exons ranging in size from 43 to 279 bp. In situ hybridisation analyses showed that mRNAs of both vap genes accumulated specifically in the subventral gland cells of H. glycines during parasitism. The gland cell-specific expression and presence of predicted secretion signal peptides in both VAPs suggest that these proteins are secreted from the nematode and may play a role in the infection of host plants by this parasite.     

立枯丝核菌(Rhizoctonia solani)对日本结缕草(Zoysia japonica steud.)的侵染过程研究

【目的】了解立枯丝核菌(Rhizoctonia solani) AG1 IA对日本结缕草(Zoysia japonica steud.)的侵染过程及其引起的病害症状,为进一步从分子水平研究该菌的病理学侵染机制和草坪草抗病分子育种提供理论基础。【方法】通过根部接种法促使立枯丝核菌与日本结缕草无菌苗建立侵染关系,从而对其病叶率、病株率及病情指数进行统计分析,同时结合组织染色透明技术及植物组织石蜡切片对立枯丝核菌的侵染过程、感染方式进行研究。【结果】立枯丝核菌AG1 IA的侵染过程主要为：菌丝吸附在植物组织表面,并沿组织表面定向生长,形成侵染结构——侵染垫与组织建立密切的侵染关系。菌丝通过细胞间隙侵入植物组织内部,主要侵染植物皮层细胞及除木质部导管以外的整个维管束系统。结缕草的地上部分与地下部分组织对立枯丝核菌的侵染显现不同的寄主反应。【结论】立枯丝核菌的侵染过程主要包括吸附、定向生长、渗透、定殖4个部分;立枯丝核菌的侵染主要引起结缕草叶片病症;结缕草病变与菌丝直接侵染无直接联系,表明该菌具有复杂的侵染机制。     

Clones of Chinese hamster cells cultivated in vitro not permanently resistant to azaguanine

The frequency of clones not permanently resistant to azaguanine (AG) was measured in Chinese hamster ovary cells (CHO) grown in vitro by plating them in 7.5 μg/ml AG and isolating a number of clones in the course of 5 experiments. Such isolated clones were propagated to a point at which their resistance to both AG and the reverse selective medium, HAT, could be determined. Out of a total of 13 clones isolated, 4 of these could not be distinguished from the parent CHO line, either on the basis of their growth in a gradient of AG concentrations or the reverse selective HAT medium or on the basis of their mutation frequency to resistance to 30 μg/ml AG. All four of the apparent phenocopies were isolated from plates in which although lower numbers of cells were seeded, a higher frequency of clones able to grow in AG was yielded. This suggests that the higher “mutation” frequencies obtained at lower cell densities are due to the appearance of phenocopies which occur only under these conditions. It is concluded that under low plating density conditions, the lower levels of AG (7.5 μg/ml) are not satisfactory for mutagenesis and mutation rate studies.     

The influence of serum components on the growth and mutation of Chinese hamster cells in medium containing 8-azaguanine

Low concentrations (?20 μg/ml) of 8-azaguanine are 1000 fold more toxic to V79 Chinese hamster cells in medium containing 10% dialyzed fetal calf serum than in medium containing 10% undialyzed serum. Serum enzyme activity that converts AG to nontoxic 8-azaxanthine degrades AG at the same rate, whether or not the serum is dialyzed. However, cytotoxicity results similar to those obtained with US were produced in medium containing DS and 2.5 μg of hypoxanthine (HX)/ml (DSH). Therefore, serum HX is considered to be responsible for the relatively low cytotoxicity of AG in medium containing US. Colonies that arose in medium containing AG were isolated and characterized. Those that remained resistant to AG (40 μg/ml) and sensitive to aminopterin in the presence of HX and thymidine (HAT) were considered mutants; nonmutants were sensitive to AG and resistant to HAT. Colonies isolated from medium containing DSH of US and low concentrations of AG were not mutants, but those from medium containing high concentrations (? μg/l) of AG were mutants. Spontaneous and N-methyl-N′-nitrosoguanidine induced mutants were detectable in medium containing DSH without replating the cells prior to adding AG (?30 μg/ml), but in order to detect MNNG induced mutations in medium containing DS replating was essential. In DS, the mutation frequency increased as an exponential function of the toxicity of MNNG, but remained two orders of magnitude lower than the induced mutation frequencies that occurred in DSH, HX, in DSH or US, produced profound effects, other than interference with AG toxicity, that distort the results of mutagenesis assays. To study mutation using AG resistance as the endpoint, it is essential to use dialyzed serum.     

Characterization of analog resistance and purine metabolism of adult rat-liver epithelial cell 8-azaguanine-resistant mutants

Adult rat-liver epithelial cultures were sensitive to the lethal effects of 8-azaguanine (AG), but lines contained variants resistant to AG. The frequency of retrievable AG-resistant colonies varied with both the concentration of AG used and the seeding density of the population under selection. Cells resistant to AG were also cross-resistant to 6-thioguanine and unable to grow in medium containing hypoxanthine, aminopterin and thymidine. Resistance was stable. AG resistance was due to a deficiency of hypoxanthine-guanine phosphoribosyl transferase (HGPRTase) activity which was not caused by an inhibitor. In the assay for HGPRTase, a substantial amount of product appeared as inosine (In) in addition to inosine monophosphate (IMP). Purine nucleoside phosphorylase will generate In from hypoxanthine and, indeed, the cells did possess this activity. However, several findings indicated that the In was derived from IMP by catabolism by 5'-nucleotidase (NTase): (1) IMP decreased as In increased and (2) the inhibitors of NTase, adenosine monophosphate and thymidine triphosphate, reduced the generation of In by over 90% without inhibiting purine nucleoside phosphorylase. The cells possessed substantial NTase activity, 35% of which was located in the cytosol along with 69% of HGPRTase. Several lines of evidence suggested that the NTase activity limited the amount of 8-azaguanylic acid presented to the cells by catabolising the nucleotide and, thereby, reducing the toxicity of available AG.     

DNA excision-repair processes in human cells can eliminate the cytotoxic and mutagenic consequences of ultraviolet irradiation

The ability of DNA excision-repair processes in diploid human fibroblasts to eliminate potentially cytotoxic and mutagenic lesions induced by UV radiation (254 nm) was demonstrated in two ways: (1) Cells with normal rates of excision were compared with cells with an intermediate rate of excision (XP2BE) and cells with an excision rate less than or equal to 1% that of normal (XP12BE) for sensitivity to the killing and mutagenic action of UV radiation. The normal cells proved resistant to doses of UV which reduced the survival of the XP cells to 14% and 0.7%, respectively, and increased the frequency of mutations to 8-azaguanine resistance in the XP cells 5- to 10-fold over background. (2) Cells in confluence were irradiated with cytotoxic and mutagenic doses of UV and allowed to carry out excision repair. After various lengths of time they were replated at lower densities to allow for expression of mutations to 6-thioguanine resistance and/or at cloning densities to assay survival. Normal cells and XP cells with reduced rates of excision repair (from complementation groups C and D) exhibited a gradual increase in survival from an initial level of 15--20% to 100% if held approximately 20 h in confluence. In contrast, XP12BE cells showed no increase from an initial survival of 20% even when held for 7 days. Normal cells irradiated in confluence but prevented from replicating for 7 days exhibited background mutation frequencies, whereas the mutation frequency in XP12BE cells did not change with the time in confluence.     

Further evidence for the genetic origin of mutations in mammalian somatic cells: the effects of ploidy level and selection stringency on dose-dependent chemical mutagenesis to purine analogue resistance in Chinese hamster ovary cells.

Whether resistance to purine analogues 8-azaguanine (AG) and 6-thioguanine (TG) in mammalian cells is due to gene mutation or to epigenetic changes was investigated by an ethyl methanesulfonate (EMS) dose-dependent induced “resistance” to these analogues in two near-diploid (2N) and one tetraploid (4N) Chinese hamster ovary (CHO) cells. EMS produced higher cell killing in 2N than in 4N cells. In the 2N cells, EMS-induced mutations to TG (1.7 μg/ml) resistance increased approximately as a linear function of the dose from 0–400 μg/ml. However, EMS was ineffective in inducing such mutation in the 4N cells. These observations are consistent with the notion that the induced TG resistance arose as a result of mutation at the gene or chromosome level. In each cell type, both the “observed” spontaneous and the EMS-induced frequency to purine analogue resistance decreased with increasing concentration of purine analogues. However, among the “resistant” clones a high proportion of those selected at 1.2 and 3.0 μg/ml of AG, a small portion selected at 7.5 μg/ml of AG, and virtually none at 1.7 and 6.0 μg/ml of TG are capable of growth in medium containing aminopterin (10 μM). This suggests that, under less stringent selective conditions, some resistant variants were being selected through mechanisms not yet defined.