Bacterial proteinases as targets for the development of second-generation antibiotics

The emergence of bacterial pathogen resistance to common antibiotics strongly supports the necessity to develop alternative mechanisms for combating drug-resistant forms of these infective organisms. Currently, few pharmaceutical companies have attempted to investigate the possibility of interrupting metabolic pathways other than those that are known to be involved in cell wall biosynthesis. In this review, we describe multiple, novel roles for bacterial proteinases during infection using, as a specific example, the enzymes from the organism Porphyromonas gingivalis, a periodontopathogen, which is known to be involved in the development and progression of periodontal disease. In this manner, we are able to justify the concept of developing synthetic inhibitors against members of this class of enzymes as potential second-generation antibiotics. Such compounds could not only prove valuable in retarding the growth and proliferation of bacterial pathogens but also lead to the use of this class of inhibitors against invasion by other infective organisms.     

RbfA, a 30S ribosomal binding factor, is a cold-shock protein whose absence triggers the cold-shock response

The cold-shock response, characterized by a specific pattern of gene expression, is induced upon a downshift in temperature and in the presence of inhibitors of ribosomal function. Here, we demonstrate that RbfA of Escherichia coli, considered to be involved in ribosomal maturation and/or initiation of translation, is a cold-shock protein. Shifting the rbfA mutant to a lower temperature resulted in a constitutive induction of the cold-shock response accompanied by slower growth at low temperatures, while shifting the rbfA mutant that overproduces wild-type RbfA resulted in an increase in total protein synthesis accompanied by faster growth adaptation to the lower temperature. Furthermore, the cold-shock response was also constitutively induced in a cold-sensitive 16S rRNA mutant at low temperatures. Accompanying the transient induction of the cold-shock response, we also report that shifting E. coli from 37°C to 15°C resulted in a temporary inhibition of initiation of translation, as evidenced by the transient decrease in polysomes accompanied by the transient increase in 70S monosomes. The accumulative data indicate that the inducing signal for the cold-shock response is the increase in the level of cold-unadapted non-translatable ribo-somes which are converted to cold-adapted translatable ribosomes by the association of cold-shock proteins such as RbfA. Therefore, the expression of the cold-shock response, and thus cellular adaptation to low temperature, is regulated at the level of translation. The data also indicate that cold-shock proteins can be translated by ribosomes under conditions that are not translatable for most mRNAs.     

Screening of inhibitors for S130G inhibitor resistant mutants of TEM type beta-lactamase

Bacteria are remarkably adaptable organisms that acquire an almost limitless competence to survive under unpleasant conditions. The drastic emergence of antibiotic resistance among β-Lactamases is the most serious threat to hospitals and nosocomial settings. β-lactam inhibitors came into existence in order to overcome the problem of antibibiotic resistance in bacteria. The emergence of inhibitor resistant mutants has raised the alarms. In this study we have used structured based virtual screening approach and have screened out some inhibitors against S130G TEM mutant. All the compounds were tested in presence and absence of conserved active site water molecules. These compounds were found be showing much higher efficacy than known β-lactamase inhibitors. Amino acids G130, S70, N132, G130, Y105 and V216 were found crucial for the interaction of inhibitors within the active site.     

Influence of the cholesterol content of mammalian spermatozoa on susceptibility to cold-shock

The cholesterol content of spermatozoa with highly unsaturated phospholipids, i.e., ram and bull, which are very susceptible to cold-shock was compared with that of rabbit and human spermatozoa which have more saturated phospholipids and a greater resistance to cold-shock. The level of cholesterol in the seminal plasma was also estimated.Cholesterol was present in ram and bull spermatozoa in comparable amounts (280–346 μg) and at approximately half the level (in micrograms per 10⁹ spermatozoa) in rabbit and human spermatozoa (545–556 μg). The molar ratio of cholesterol: phospholipid in rabbit and human spermatozoa was 0.88 and 0.99, respectively, which is similar to the ratio of these lipids in erythrocytes and higher than the ratio of 0.38 and 0.45 for ram and bull spermatozoa, respectively. This ratio is of some importance in determining the nature and degree of packing in the spermatozoan membrane; higher levels of cholesterol result in a more cohesive, rigid, and impermeable structure. A definite relationship is apparent between the ratio of cholesterol: phospholipid, the ratio of polyunsaturated: saturated phospholipid-bound fatty acids, and the susceptibility of the spermatozoa to cold-shock.     

Theoretical aspects of enzyme induction and inhibition leading to the reversal of resistance to biocides

The well-known phenomena of enzyme induction and inhibition have been applied in the enunciation of two mechanisms which could be used in the reversal of resistance which organisms develop towards biocides (drugs and pesticides) in many cases. For those biocides active per se which are metabolized by inducible enzymes to non-toxic metabolites, resistant organisms would be those possessing high levels of drug-metabolizing enzymes (Mechanism 1). For those biocides inactive per se but requiring metabolic activation for activity, resistant organisms would be those possessing low levels of drug metabolizing enzymes (Mechanism 2). In mechanism 1, the addition of enzyme inhibitors to the biocide would be effective in reversing resistance. In mechanism 2 the addition of an enzyme inducer to the biocide would increase the susceptibility of the resistant organisms. An ectoparasite insecticide 2-chloro-1-(2,4 dichlorophenyl) vinyl diethylphosphate (chlorfenvinphos or supona) is used as an example for mechanism 1. The malarial drugs primaquine and chloroquine are used as examples of mechanism 2.     

Natural resistance to inhibitors of the ubiquinol cytochrome c oxidoreductase of Rubrivivax gelatinosus: sequence and functional analysis of the cytochrome bc(1) complex

Biochemical analyses of Rubrivivax gelatinosus membranes have revealed that the cytochrome bc(1) complex is highly resistant to classical inhibitors including myxothiazol, stigmatellin, and antimycin. This is the first report of a strain exhibiting resistance to inhibitors of both catalytic Q(0) and Q(i) sites. Because the resistance to cytochrome bc(1) inhibitors is primarily related to the cytochrome b primary structure, the petABC operon encoding the subunits of the cytochrome bc(1) complex of Rubrivivax gelatinosus was sequenced. In addition to homologies to the corresponding proteins from other organisms, the deduced amino acid sequence of the cytochrome b polypeptide shows (i) an E303V substitution in the highly conserved PEWY loop involved in quinol/stigmatellin binding, (ii) other substitutions that could be involved in resistance to cytochrome bc(1) inhibitors, and (iii) 14 residues instead of 13 between the histidines in helix IV that likely serve as the second axial ligand to the b(H) and b(L) hemes, respectively. These characteristics imply different functional properties of the cytochrome bc(1) complex of this bacterium. The consequences of these structural features for the resistance to inhibitors and for the properties of R. gelatinosus cytochrome bc(1) are discussed with reference to the structure and function of the cytochrome bc(1) complexes from other organisms.     

Differential inhibition of mollicute growth: an approach to development of selective media for specific mollicutes

The energy-generating pathways of Mycoplasma spp. are diverse. Thus, it was predicted that the ability of inhibitors of these pathways to block growth would vary among species. This prediction was tested with 14 Mycoplasma species and potential inhibitors. The greatest differentiation among test species was obtained using fluoride, iodoacetate (IAA), beta-fluoropyruvate (FP), cibacron blue (CB), L-citrulline, and carbonyl cyanide m-chlorophenylhydrazone. A range of other potential inhibitors, including L-arginine analogues, had little inhibitory effect on growth, and D-arginine was shown to be a growth substrate for arginine-hydrolyzing species. Fluoride selectively inhibited the growth of mycoplasmas that were able only to ferment sugars to lactate and/or to hydrolyze arginine. In contrast, IAA was most effective against organic acid-oxidizing species, and L-citrulline inhibited arginine-hydrolyzing species. Mycoplasma verecundum, a species for which energy sources have not been identified, was relatively resistant to FP. Similarly, Acholeplasma laidlawii was distinguished by its CB resistance.     

Characterization of Cold-Shock Protein A of Antarctic <Emphasis Type="Italic">Streptomyces</Emphasis> sp. AA8321

Abstact Polar organisms should have mechanisms to survive the extremely cold environment. Four genes encoding cold-shock proteins, which are small, cold-induced bacterial proteins, have been cloned from the Antarctic bacterium Streptomyces sp. AA8321. Since the specific functions of any polar bacterial or Streptomyces cold-shock proteins have not yet been determined, we examined the role of cold-shock protein A from Streptomyces sp. AA8321 (CspA_St). Gel filtration chromatography showed that purified CspA_St exists as a homodimer under physiological conditions, and gel shift assays showed that it binds to single-stranded, but not double-stranded, DNA. Overexpression of CspA_St in Escherichia coli severely impaired the ability of the host cells to form colonies, and the cells developed an elongated morphology. Incorporation of a deoxynucleoside analogue, 5-bromo-2′-deoxyuridine, into newly synthesized DNA was also drastically diminished in CspA_St-overexpressing cells. These results suggest that CspA_St play a role in inhibition of DNA replication during cold-adaptation.     

Larval temperature experience determines sensitivity to cold-shock-induced wing color pattern changes in the blue pansy butterfly Junonia orithya

The butterfly wing color patterns are unique to a species but are modified in response to cold-shock and tungstate treatments at the pupal stage, producing characteristic temperature–shock (TS) phenotypes that are distinct from the color patterns of seasonal polyphenism. In this study, we examined the efficiency of cold-shock and tungstate treatments for color pattern modifications at the pupal stage in relation to larval rearing conditions for the fall or summer morph using the blue pansy butterfly Junonia orithya. We found that larvae reared under the low-temperature condition that induces the fall morph exhibited hardiness against the color pattern changes imposed by cold-shock or tungstate treatment at the pupal stage. When larvae were fed an artificial diet containing tungstate under the high-temperature condition that induces the summer morph, they were still vulnerable to color pattern changes imposed by cold-shock or tungstate treatment at the pupal stage. Furthermore, larvae reared under the high-temperature condition were subjected to cold-shock or tungstate treatments at the pupal stage. In addition to the expected TS-type changes, these individuals exhibited a reduced number of eyespots in adults, which is a feature of the fall morph. These results suggest that the temperature condition experienced by the larvae, but not their consumption of tungstate, determines the sensitivity of the wing imaginal discs to cold-shock and tungstate treatments at the pupal stage.     

Inhibitors of retrovirus infection are secreted by several hamster cell lines and are also present in hamster sera.

We have previously shown that Chinese hamster ovary (CHO) cells are resistant to infection by gibbon ape leukemia virus and amphotropic pseudotype retroviral vectors because of the secretion of factors that inhibit retrovirus infection. Such factors were not secreted by any mouse or human cell lines tested. Secretion of the inhibitors and resistance to infection are abrogated by treatment of CHO cells with the glycosylation inhibitor tunicamycin. Here we show that the inhibitory activities against gibbon ape leukemia virus and amphotropic viruses are partially separable and that glycosylation mutations in CHO cells mimic the effects of tunicamycin treatment. We find that several hamster cell lines derived from both Chinese and Syrian hamsters secrete inhibitors of retrovirus infection, showing that these inhibitors are not unique to the CHO cell line. Inhibitory factors are also present in the sera of Chinese and Syrian hamsters but were not detected in bovine serum. These results suggest the presence of specific factors that function to inhibit retrovirus infection in hamsters.     

Semen plasma proteins prevent cold-shock membrane damage to ram spermatozoa

Although the effect of semen plasma on the function of spermatozoa has been widely studied, results are contradictory. We showed that semen plasma proteins are adsorbed onto the cold-shocked ram sperm surface, and that this adsorption is able to reverse the membrane alterations induced by cold-shock. In the present study we evaluate whether the addition of semen plasma proteins before the cold-shock would prevent membrane damage and maintain ram sperm viability. Ram spermatozoa freed from semen plasma by a dextran/swim-up procedure were strongly affected by the cold-shock treatment, lowering cell viability (membrane integrity by fluorescence markers) from 72.2+/-3.4% to 24.6+/-2.1%. Adding semen plasma proteins (> 3 kDa) to the medium before the cold treatment had an immediate beneficial effect on sperm survival in all samples. This effect was concentration-dependent, since the percentage of membrane-intact spermatozoa increased significantly with increased protein concentration in the incubation medium. The highest concentration of proteins (2.1 mg) continued to protect the membranes after 1 h of incubation at 20 degrees C while lower concentrations (0.7 and 1.4 mg) showed a slight decline. Inclusion of linoleic-oleic acids had a beneficial effect on preserving sperm viability when 25, 37 or 75 microM linoleic-oleic acids were added. There was a positive interaction between fatty acids and semen plasma proteins. Thus, the addition of 25 microM oleic-linoleic acid in the presence of 2.1 mg semen plasma proteins accounted for an increase in viability up to 50.7% significance (P < 0.001) relative to the control sample (25%). Likewise, semen plasma proteins significantly promoted the ability of Vitamin E (alpha-tocopherol phosphate) to improve sperm survival. A 26% viability value obtained after cold-shock in the control sample significantly increased (P < 0.001) up to 57% in the sample with 1.6 mM Vitamin E phosphate and 2.1 mg semen plasma proteins (0 h). This study demonstrates that impaired function of cold-shocked ram spermatozoa freed from semen plasma could be prevented by addition of semen plasma proteins, resulting in higher maintained viability values. Inclusion of either linoleic-oleic acids or vitamin E together with semen plasma proteins would increase the improvement in ram spermatozoa survival.     

绿脓杆菌群集生长效应

本文通过群集的绿脓杆菌在高盐、酸性培养基上生长试验,抗金黄色葡萄球菌的拮抗试验及冷休克试验,证明了绿脓杆菌群集生长效应。主要表现为群集的绿脓杆菌可以在含10～20%NaCl 的培养基上生长;pH4环境中也可生长。同样条件下,对照几乎均为阴性。分散成单个细胞分布的绿脓杆菌对冷反应敏感,群集则有抗冷休克作用。实验条件下,密集的金黄色葡萄球菌生长对分散的绿脓杆菌有拮抗作用,群集则可抵抗这种作用而生长。讨论中初步分析了群集生长效应的机理和对细菌种群在自然生境中的稳定作用。     

The impact of transposable elements on eukaryotic genomes: From genome size increase to genetic adaptation to stressful environments

Transposable elements (TEs) are present in roughly all genomes. These mobile DNA sequences are able to invade genomes and their impact on genome evolution is substantial. The mobility of TEs can induce the appearance of deleterious mutations, gene disruption and chromosome rearrangements, but transposition activity also has positive aspects and the mutational activities of TEs contribute to the genetic diversity of organisms. This short review aims to give a brief overview of the impact TEs may have on animal and plant genome structure and expression, and the relationship between TEs and the stress response of organisms, including insecticide resistance.     

Non-methylene-interrupted fatty acids from marine invertebrates: Occurrence,characterization and biological properties

Marine organisms, in particular invertebrates, have proved to be a major source of unique fatty acid (FA) structures originating from unusual biosynthetic pathways. Among them, non-methylene-interrupted (NMI) FA occur in various molluscs in the wide ranges of concentrations (up to 20%), such as the most often encountered 20:2 Δ5,11, 20:2 Δ5,13, 22:2 Δ7,13 or 22:2 Δ7,15. Such NMI FA have also been reported from algae, echinoderms, sponges, tropical rays, and many other invertebrates. The most intriguing marine invertebrates seem to be sponges that commonly contain very long-chain Δ5,9 FA. A third double bond can occur in the NMI FA as reported in some marine organisms, such as 20:3 Δ7,13,16 or 30:3 Δ5,9,23. Lipids of invertebrates from deep-sea hydrothermal and cold-seep vents gave rise to an intense research activity including reports on unprecedented NMI polyunsaturated FA. The bivalve molluscs are able to synthesize de novo the NMI FA but their precise biological interest is presently not well-known, although structural and functional roles in biological membranes have been suggested, in particular a higher resistance to oxidative processes and microbial lipases. Biosynthetic pathways of Δ5,9 FA in sponges were demonstrated up to C₂₆ FA structures and include particular elongation and desaturation steps. Recently, intense research effort has been conducted to investigate the biomedical potential of these unusual FA. Thus, Δ5,9 FA displayed interesting antiplasmodial activity. The most promising FA topoisomerase I inhibitors to date seem to be the long-chain Δ5,9 FA. This inhibitory activity is probably partially responsible for the toxicity displayed by some of the Δ5,9 FA towards cancer cell lines.     

高温胁迫下番茄幼苗对冷激的响应

高温逆境是影响夏秋季蔬菜设施集约化育苗质量的主要因素之一,利用温度逆境诱导植物产生交叉适应是植物获得抗逆性的一种有效手段.为探索冷激强度对番茄幼苗高温胁迫的缓解效应,试验采用人工气候箱模拟夏季设施中的高温胁迫,研究了不同冷激温度(5、10、15 ℃)和冷激持续时间(10、20、30 min)对番茄幼苗生长、生物膜保护系统的影响,并研究了单次适宜冷激处理对番茄小分子热激蛋白LeHSP23.8和CaHSP18基因表达的影响.结果表明： 在高温胁迫前对番茄幼苗进行冷激处理可以抑制其下胚轴的伸长和株高的生长.冷激缓解番茄幼苗高温胁迫的效应在不同冷激温度下表现出不同的变化趋势;5 ℃冷激处理抑制了番茄幼苗抗氧化酶活性的升高,使细胞膜透性增大,对幼苗产生伤害;10 ℃冷激处理对番茄幼苗高温胁迫的缓解效应随冷激时间的延长呈降低趋势;而15 ℃冷激处理缓解番茄幼苗高温胁迫的效应随冷激时间的延长呈增加趋势.适宜冷激温度和冷激持续时间能够诱导番茄幼苗对高温逆境的交叉适应性,在高温胁迫前将番茄幼苗进行温度为10 ℃、持续10 min的冷激处理效果最佳,与对照相比,显著提高了高温胁迫下番茄幼苗植株单株干质量和壮苗指数,降低了番茄幼苗叶片相对电导率和丙二醛含量,促进了脯氨酸和可溶性蛋白的积累,提高了番茄幼苗叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT) 3种抗氧化酶活性,并诱导了小分子热激蛋白基因LeHSP23.8和CaHSP18在常温条件下的上调表达.
     

Cost of resistance to parasites in digital organisms

Virtually all organisms are attacked by parasites and are therefore expected to evolve resistance against these natural enemies. Parasite resistance is costly in a wide range of organisms, although the generality of such costs has been questioned, especially when resistance is not based on reallocation of resources. Digital organisms are increasingly used to explore aspects of life in general. In the Tierra system, there is a trade-off between resistance against parasites and competitive ability. Because digital organisms are too simple to store resources, the finding that resistance to parasites is costly in digital organisms suggests that costs of parasite resistance can also occur when resistance is not resource based.