In Vitro and In Vivo Characterization of Pyocin

Pyocin, a bacteriocin obtained from lysates of ultraviolet-induced cultures of Pseudomonas aeruginosa was characterized in vitro and in vivo after 1,000-fold purification by chemical, column, and differential centrifugation procedures. Electron micrographs of negatively stained pyocin preparations contained rod-shaped particles which resembled the contractile tail protein of the T-even phages of Escherichia coli. Although two separate and distinct pyocin fractions were eluted from diethylaminoethyl cellulose (pH 7.5) during the purification procedure, the particles appeared identical. In addition, the two fractions exhibited a close correlation between their titers and the particle numbers as observed in the electron microscope. The particles were approximately 20 by 90 mmu with a core diameter of 5 mmu and a sheath length of 50 mmu. Neither intact phage nor ghosts were seen in any of the preparations, although ringlets of two different diameters, which appeared to correspond to the diameters of the sheath and inner core, were observed. Other studies indicated that, although crude preparations were stable to freezing and thawing, purified preparations lost all of their activity under similar treatment. However, the addition of 50% glycerol to purified preparations completely protected activity. Conversely, aged normal human or rabbit sera enhanced the antibacterial activity of pyocin approximately fourfold, although serum albumin and hemoglobin had no effect. In vivo studies indicated that purified pyocin was not lethal for mice when injected intraperitoneally in concentrations of 28,000 to 1,400,000 units (5.6 to 276 mug of protein), nor was 7,200 to 36,000 units dermonecrotic for rabbits.     

Regulation of Pyruvate Decarboxylase In Vitro and In Vivo

Results presented in this paper strongly support the view thatregulation of the key enzyme of alcoholic fermentation, pyruvatedecarboxylase (PDC), is achieved in a number of ways, all associatedwith possible lowering of the cytoplasmic pH during anoxia.These mechanisms include not only the well-known acid pH optimumof PDC, but also long-term, reversible changes in characteristicsof the enzyme established both in vitro and in vivo. Following transfer of desalted extracts from pH 6.0 to 7.4,maximal activity of PDC was decreased, while there was a considerableincrease in the lag before maximal activity was reached. Similarchanges in enzyme characteristics were observed when wheat (Triticumaestivum L. cv. Gamenya) roots and rice (Oryza sativa L. cv.Calrose) coleoptiles were transferred from anoxic to aerobicsolutions, provided PDC was assayed within 10 min of the startof maceration. All of the above changes were usually readilyreversible when extracts were returned to pH 6.0, or when plantswere returned to anoxic solutions. Additional regulation of PDC would be achieved by the S_0.5 forpyruvate which is 0.75 mol m^–3 at pH 6.0, 1.0 mol m^–3at pH 6.8, and 2.5 mol m^–3 at pH 7.4; the latter is wellabove estimates for pyruvate concentrations in the cytoplasmof aerated tissues. We assess that the combined effects of the acid pH optimum,the high S_0.5 at pH 7.4 and the long-term decreases in activityobserved during incubation at pH 7.4 would reduce PDC activityin aerobic cells to at most 7% of the activity in anoxic cells.Possible additional controls for the pathway of alcoholic fermentationare briefly considered. Key words: PDC, regulation, anoxia     

In Vitro and In Vivo Studies of Streptomycin-Dependent Cholera Vibrios

Streptomycin-dependent cholera vibrio strains were derived from Inaba, Ogawa, and NAG vibrios by the method of Mel. These phenotypes grew more slowly and attacked fermentable substances after a longer period of time than the streptomycin-sensitive parent strains. Rabbits injected with streptomycin-sensitive strains and their streptomycin-dependent forms showed homologous agglutinin production. Patas monkeys fed with 10(9) streptomycin-dependent strains shed them for 1 to 2 days without ill effect, whereas the same number of streptomycin-independent organisms caused disease. The possibility of the application of multiple doses of streptomycin-dependent organisms in oral immunization against cholera was considered.     

In Vitro and In Vivo Characterization of the Alkaloid Nuciferine

Rationale

The sacred lotus (Nelumbo nucifera) contains many phytochemicals and has a history of human use. To determine which compounds may be responsible for reported psychotropic effects, we used in silico predictions of the identified phytochemicals. Nuciferine, an alkaloid component of Nelumbo nucifera and Nymphaea caerulea, had a predicted molecular profile similar to antipsychotic compounds. Our study characterizes nuciferine using in vitro and in vivo pharmacological assays.

Methods

Nuciferine was first characterized in silico using the similarity ensemble approach, and was followed by further characterization and validation using the Psychoactive Drug Screening Program of the National Institute of Mental Health. Nuciferine was then tested in vivo in the head-twitch response, pre-pulse inhibition, hyperlocomotor activity, and drug discrimination paradigms.

Results

Nuciferine shares a receptor profile similar to aripiprazole-like antipsychotic drugs. Nuciferine was an antagonist at 5-HT_2A, 5-HT_2C, and 5-HT_2B, an inverse agonist at 5-HT₇, a partial agonist at D_2, D₅ and 5-HT₆, an agonist at 5-HT_1A and D₄ receptors, and inhibited the dopamine transporter. In rodent models relevant to antipsychotic drug action, nuciferine blocked head-twitch responses and discriminative stimulus effects of a 5-HT_2A agonist, substituted for clozapine discriminative stimulus, enhanced amphetamine induced locomotor activity, inhibited phencyclidine (PCP)-induced locomotor activity, and rescued PCP-induced disruption of prepulse inhibition without induction of catalepsy.

Conclusions

The molecular profile of nuciferine was similar but not identical to that shared with several approved antipsychotic drugs suggesting that nuciferine has atypical antipsychotic-like actions.     

Interferon Production by Human Cells In Vitro

The relative capacity of several types of human cells and tissue to produce interferon was studied. Types of cells and tissue included were fibroblasts from embryos, foreskins, and biopsied skins; amnion cells; peripheral leukocytes; established lymphoid cell lines; established heteroploid cell lines; and chorioamniotic membrane. When Newcastle disease virus was used as the inducer, fibroblasts and amnion cells produced more interferon per 10⁶ cells than leukocytes, lymphoid cells, and heteroploid cells. Only minor variations in interferon-producing capacity were observed among fibroblasts from 36 persons. Culture passage level, cell concentration, and inducer were factors that significantly affected interferon production.     

In Vitro and In Vivo Isolation and Characterization of Duvenhage Virus

A fatal human case of Duvenhage virus (DUVV) infection in a Dutch traveller who had returned from Kenya was reported in 2007. She exhibited classical symptoms of rabies encephalitis with distinct pathological findings. In the present study we describe the isolation and characterization of DUVV in vitro and its passage in BALB/c mice. The virus proved to be neuroinvasive in both juvenile and adult mice, resulting in about 50% lethality upon peripheral infection. Clinical signs in infected mice were those of classical rabies. However, the distribution of viral antigen expression in the brain differed from that of classical rabies virus infection and neither inclusion bodies nor neuronal necrosis were observed. This is the first study to describe the in vitro and in vivo isolation and characterization of DUVV.     

一种体内标记转录RNA的新技术

目的:验证一种体内标记转录RNA的新技术,并用该技术观察一种长寿药物雷帕霉素对真核细胞HEK293中RNA合成的影响。方法:将尿嘧啶类似物5-乙炔尿苷(EU)和雷帕霉素加到HEK293细胞培养基中,共同孵育2h,然后在激光共聚焦显微镜下对EU标记的新合成RNA进行观察;用Image-pro plus软件对图像的荧光强度进行分析,获得反应荧光强度的平均光密度数值;用SPSS软件对数值进行统计分析。结果:激光共聚焦显微镜下,可见EU标记的新合成RNA主要分布在胞质和核仁中,以核仁中荧光最强;Image-pro plus软件和SPSS软件分析表明,加入雷帕霉素前后,细胞新合成的RNA平均光密度无明显差别。结论:EU是一种安全、简单、快速而灵敏的检测新合成RNA的新技术,用该技术检测表明长寿药物雷帕霉素不影响真核细胞HEK293中总RNA的合成。     

A Convenient Method for Labeling Oligonucleotide Analogs by a Fluorogenic Tag for Direct In Vitro and In Vivo Visualization of Carboxyesterase Activity

Abstract

The synthesis of two oligonucleoside phosphorothioate and methyl-phosphonate analogs functionalized with a fluorogenic tag is described. The fluorescent conjugate formation is demonstrated in cell culture medium, cell extracts and in human fibroblasts.     

Selective Pressures on RNA Hairpins In Vivo and In Vitro

Comparison of the most stable potential hairpins in the sequences of natural ribozymes with those in the randomized sequences has revealed that the hairpin loop energies are lower than expected by chance. Although these hairpins are not necessarily parts of functional structures, there is a selective pressure to diminish the destabilizing free energies of the hairpin loops. In contrast, no significant bias is observed in the stacking values of the most stable stems. In the ribozymes isolated in vitro the loops of potential hairpins are closer to random values, which can result in less efficient folding rates. Furthermore, the effects of kinetic traps seem to be more significant in the folding pathways of the in vitro isolates due to a potential to form stable stacks incompatible with the functional folds. Similarly to natural ribozyme sequences, the untranslated regions of viral RNAs also form hairpins with relatively low loop free energies. These evolutionary trends suggest ways for efficient engineering of improved RNA constructs on the basis of analysis of in vitro isolates and approaches for the search of regions coding for functional RNA structures in large genome sequences. Received: 12 January 2001 / Accepted: 21 May 2001     

In Vitro and In Vivo Laboratory Evaluation of Cephaloglycin and Cephaloridine

Two new antibiotics, structurally related to cephalothin, have been given the generic names cephaloglycin and cephaloridine. Cephaloglycin is the dipolar ion of 7-(d-α-aminophenylacetamido)-cephalosporanic acid. Cephaloridine is 7-[α-(2-thiophene)acetamido]-3-(1-pyridylmethyl)-3-cephem-4-carboxylic acid betaine. These new compounds were evaluated simultaneously. The broad spectrum of activity observed in vitro and in vivo with both antibiotics, the good oral absorption obtained with cephaloglycin, and the stability of cephaloridine are emphasized. The data suggest that both antibiotics merit clinical trial in humans.     

Cytomegalovirus Replicon-Based Regulation of Gene Expression In Vitro and In Vivo

There is increasing evidence for a connection between DNA replication and the expression of adjacent genes. Therefore, this study addressed the question of whether a herpesvirus origin of replication can be used to activate or increase the expression of adjacent genes. Cell lines carrying an episomal vector, in which reporter genes are linked to the murine cytomegalovirus (MCMV) origin of lytic replication (oriLyt), were constructed. Reporter gene expression was silenced by a histone-deacetylase-dependent mechanism, but was resolved upon lytic infection with MCMV. Replication of the episome was observed subsequent to infection, leading to the induction of gene expression by more than 1000-fold. oriLyt-based regulation thus provided a unique opportunity for virus-induced conditional gene expression without the need for an additional induction mechanism. This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV. Moreover, the application of this principle for intracellular immunization against herpesvirus infection was demonstrated. The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth. This conditional system was operative in explant cultures of transgenic mice, but not in vivo. Several applications are discussed.     

Endothelial Dysfunction of Bypass Graft: Direct Comparison of In Vitro and In Vivo Models of Ischemia-Reperfusion Injury

Background

Although, ischemia/reperfusion induced vascular dysfunction has been widely described, no comparative study of in vivo- and in vitro-models exist. In this study, we provide a direct comparison between models (A) ischemic storage and in-vitro reoxygenation (B) ischemic storage and in vitro reperfusion (C) ischemic storage and in-vivo reperfusion.

Methods and Results

Aortic arches from rats were stored for 2 hours in saline. Arches were then (A) in vitro reoxygenated (B) in vitro incubated in hypochlorite for 30 minutes (C) in vivo reperfused after heterotransplantation (2, 24 hours and 7 days reperfusion). Endothelium-dependent and independent vasorelaxations were assessed in organ bath. DNA strand breaks were assessed by TUNEL-method, mRNA expressions (caspase-3, bax, bcl-2, eNOS) by quantitative real-time PCR, proteins by Western blot analysis and the expression of CD-31 by immunochemistry. Endothelium-dependent maximal relaxation was drastically reduced in the in-vivo models compared to ischemic storage and in-vitro reperfusion group, and no difference showed between ischemic storage and control group. CD31-staining showed significantly lower endothelium surface ratio in-vivo, which correlated with TUNEL-positive ratio. Increased mRNA and protein levels of pro- and anti-apoptotic gens indicated a significantly higher damage in the in-vivo models.

Conclusion

Even short-period of ischemia induces severe endothelial damage (in-vivo reperfusion model). In-vitro models of ischemia-reperfusion injury can be limitedly suited for reliable investigations. Time course of endothelial stunning is also described.     

In Vitro and In Vivo Activity of Hamycin Against Blastomyces dermatitidis

Clinical responses of patients with blastomycosis to treatment with hamycin have been variable. An explanation for this was sought in a series of studies in which in vitro and in vivo susceptibilities to hamycin of five strains of Blastomyces dermatitidis were compared. Minimal inhibitory concentrations of hamycin for the five strains indicated uniformly high levels of in vitro susceptibility (0.008 to 0.016 μg/ml). In vivo activity was measured in infected mice treated intraperitoneally for a period of 28 days with doses of the drug ranging from 0.001 to 0.030 mg per mouse. Significant differences in response to treatment among the five strains were noted (P < 0.001), and protective doses were found to vary from 0.001 to >0.030 mg per mouse per day. Further observations of infected mice after treatment revealed marked rates of relapsing infection, and several strains caused death. Persistent inapparent infections were also detected on culture of selected organs. Toxicity due to hamycin alone was not observed. These results suggest that variations in clinical responses to hamycin therapy in treatment of blastomycosis reflect differences in pathogenesis and host response in vivo to the infecting organism rather than differences in susceptibility of B. dermatitidis to hamycin.     

Inhibition of Sphingolipid Synthesis by Cycloserine In Vitro and In Vivo

Abstract: d - and l -cycloserine were shown to be irreversible inhibitors of the first enzyme of the sphingolipid pathway, 3-ketodihydrosphingosine synthetase, in a study using bacterial and brain microsomal enzymes, l-Cycloserine was shown to be 100 times more inhibitory than the d-isomer for the brain microsomal enzyme in vitro. In vivo , l -cycloserine caused a 70% inhibition of brain microsomal enzyme. Following one injection, enzyme activity recovered 80% of normal after 16 hours. Daily dosages of l -cycloserine on a regimen of intraper-itoneal injection for 7 days caused a significant reduction in total brain ganglioside and cerebroside plus sulfatide levels.     

In Vitro and In Vivo Evaluations of PLGA Microspheres Containing Nalmefene

Poor patient compliance, untoward reactions and unstable blood drug levels after the bolus administration are impeding the pharmacotherapy for insobriety. A long-acting preparation may address these limitations. The aim of this paper was to further investigate the in vitro characteristics and in vivo performances of nalmefene microspheres. Nalmefene was blended with poly (lactide-co-glycolide) (PLGA) to prepare the target microspheres by an O/O emulsification solvent evaporation method. The prepared microspheres exhibited a controlled release profile of nalmefene in vitro over 4 weeks, which was well fitted with a first-order model. In vitro degradation study showed that the drug release in vitro was dominated by both drug diffusion and polymer degradation mechanisms. Pharmacokinetics study indicated that the prepared microspheres could provide a relatively constant of nalmefene plasma concentration for at least one month in rats. The in vivo pharmacokinetics profile was well correlated with the in vitro drug release. Pharmacodynamics studies revealed that the drug loaded microspheres could produce a long-acting antagonism efficacy on rats. These results demonstrated the promising application of injectable PLGA microspheres containing nalmefene for the long-term treatment of alcohol dependence.