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     

Antithrombotic Activities of Luteolin In Vitro and In Vivo

The objectives of present study were to investigate whether luteolin affects procoagulant proteinase activity and fibrin clot formation and influences thrombosis and coagulation in Sprague–Dawle rats. Luteolin significantly inhibited the enzymatic activity of thrombin and FXa activity by 29.1% and 16.2%. Luteolin also inhibited fibrin polymer formation in turbidity and microscopic analysis using fluorescent conjugate. Coagulation assay of luteolin was found to prolong activated partial thromboplastin time and prothrombin time. Moreover, luteolin protected the development of oxidative stress induced thrombosis in the FeCl₃‐induced carotid arterial thrombus model. This study demonstrated that luteolin may be useful by reducing or preventing thrombotic challenge and can help us better understand the antithrombotic action of luteolin.     

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.     

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.     

Distant Homology Modeling of LCAT and Its Validation through In Silico Targeting and In Vitro and In Vivo Assays

LCAT (lecithin:cholesterol acyltransferase) catalyzes the transacylation of a fatty acid of lecithin to cholesterol, generating a cholesteryl ester and lysolecithin. The knowledge of LCAT atomic structure and the identification of the amino acids relevant in controlling its structure and function are expected to be very helpful to understand the enzyme catalytic mechanism, as involved in HDL cholesterol metabolism. However - after an early report in the late ‘90 s - no recent advance has been made about LCAT three-dimensional structure. In this paper, we propose an LCAT atomistic model, built following the most up-to-date molecular modeling approaches, and exploiting newly solved crystallographic structures. LCAT shows the typical folding of the α/β hydrolase superfamily, and its topology is characterized by a combination of α-helices covering a central 7-strand β-sheet. LCAT presents a Ser/Asp/His catalytic triad with a peculiar geometry, which is shared with such other enzyme classes as lipases, proteases and esterases. Our proposed model was validated through different approaches. We evaluated the impact on LCAT structure of some point mutations close to the enzyme active site (Lys218Asn, Thr274Ala, Thr274Ile) and explained, at a molecular level, their phenotypic effects. Furthermore, we devised some LCAT modulators either designed through a de novo strategy or identified through a virtual high-throughput screening pipeline. The tested compounds were proven to be potent inhibitors of the enzyme activity.     

In Vivo,In Vitro,and In Silico Characterization of Peptoids as Antimicrobial Agents

Bacterial resistance to conventional antibiotics is a global threat that has spurred the development of antimicrobial peptides (AMPs) and their mimetics as novel anti-infective agents. While the bioavailability of AMPs is often reduced due to protease activity, the non-natural structure of AMP mimetics renders them robust to proteolytic degradation, thus offering a distinct advantage for their clinical application. We explore the therapeutic potential of N-substituted glycines, or peptoids, as AMP mimics using a multi-faceted approach that includes in silico, in vitro, and in vivo techniques. We report a new QSAR model that we developed based on 27 diverse peptoid sequences, which accurately correlates antimicrobial peptoid structure with antimicrobial activity. We have identified a number of peptoids that have potent, broad-spectrum in vitro activity against multi-drug resistant bacterial strains. Lastly, using a murine model of invasive S. aureus infection, we demonstrate that one of the best candidate peptoids at 4 mg/kg significantly reduces with a two-log order the bacterial counts compared with saline-treated controls. Taken together, our results demonstrate the promising therapeutic potential of peptoids as antimicrobial agents.     

Quantitative Differences In Phagocytosis and Degradation of Pneumocystis By Alveolar Macrophages In Aids and Non-Hiv Patients In Vivo

Broncholaveolar lavage (BAL) specimens (n= 213) from AIDS and non-HIV immunosuppressed patients were investigated for the presence of Pneumocystis carinii infection by fluorescence microscopy of Papanicolaou-stained slides. Alveolar casts, extracellular pneumocysts and phagocytosed cysts and their degradation products in pulmonary alveolar macrophages were identified. the number of phagocytosed pneumocysts within human pulmonary alveolar macrophages was recorded and correlated with the number of extracellular cysts and alveolar casts, in both groups of patients. Both phagocytic and degradation capacity were depressed in AIDS patients. This observation may explain the large number of extracellular organisms found in BAL specimens of AIDS patients compared with non-HIV-positive immunocompromised individuals.     

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.     

Mitotic and Labelling Activity In Normal Human Epidermis In Vivo

Labelling and mitotic indices were studied in the epidermis of twenty-eight young men. A mean labelling index of 5.5% was found from the whole study and a mean mitotic index of 0.06%. Mitotic index particularly was extremely variable; indices between 0.002 and 0.438% were found in individual biopsies. In the first two of three experiments in which mitotic index at 09.00 hours was compared with that at 15.00 hours, significant differences were found (15.00 hours > 09.00 hours by a factor of 2.6, P < 0.001). However, in the third such experiment no such difference was found, suggesting that the timing and occurrence of diurnal rhythms of mitotic activity may not be consistent in normal human epidermis. In the one experiment in which it was investigated, a significantly higher mitotic index was found at 21.00 hours compared to 09.00 and 15.00 hours. Labelling index did not vary significantly at 09.00, 15.00 or 21.00 hours. However, labelling index did show a significant pattern of change over a 12-month period in two groups of subjects; peaks of labelling were seen in July and troughs in January. Very high ratios of labelled: mitotic cells were found, the median ratio for the whole study being ninety-eight labelled: one mitotic cell. This finding supports the possibility that not all labelled cells subsequently go on to divide in normal human epidermis.     

Dysmyelination of Shiverer Mutant Mice In Vivo and In Vitro

Demyelination in the CNS of shiverer mutant mice was studied in vivo and in vitro. By immunohistochemical reaction with glial fibrillary acidic protein antibody, hypertrophy of the fibrous astrocytes was observed in the white matter of shiverer cerebella. The cerebella of shiverer mice in primary culture from the day of birth showed very poor myelination under optical microscopy. Axons of Purkinje cells are thought to be the main myelinated axons in the primary culture of the cerebellum. Purkinje cells from shiverer appeared normal with regard to Bodian silver impregnation, hematoxylin and eosin staining, and P400 protein characterization of Purkinje cells. Addition of the conditioned culture medium of shiverer to the control culture did not interfere with myelination. We concluded that the demyelination in the CNS of shiverer could be caused by an intrinsic defect of the oligodendrocyte rather than by hypertrophy of the astrocytes or by diffusible factors.     

In Vivo and In Vitro Action of Norethindrone on Staphylococci

Norethindrone has been examined in vitro for antibacterial activity against 10 microorganisms. Turbidimetric techniques were used to assay the antibacterial activity of norethindrone. The organisms tested included Staphylococcus aureus, S. epidermidis, Micrococcus conglomeratus, Listeria monocytogenes, Streptococcus faecalis, Salmonella typhosa, Shigella flexnerii, Klebsiella pneumoniae, Escherichia coli, and Proteus vulgaris. Bacteriostatic action was shown only against the gram-positive microorganisms when they were grown anaerobically in Tryptic Soy Broth containing 10 to 50 mug of norethindrone per ml. The bacteriostatic action of norethindrone was exerted primarily during the first 8 hr of incubation and it was reduced by the presence of oxygen. Mestranol at a concentration of 1 to 10 mug/ml failed to exert any significant action on S. aureus. However, incorporation of 5 mug of mestranol per ml in the culture medium enhanced the bacteriostatic action of norethindrone on staphylococci. Enhancement of the bacteriostatic action of norethindrone could not be obtained by the addition of a concentration of 5 mug/ml of testosterone, 17alpha-estradiol, and 17beta-estradiol. Progesterone and 4-pregnen-20beta-ol-3-one under similar conditions showed an additive bacteriostatic effect when they were incorporated into the culture medium containing norethindrone. In vivo studies indicated that female, adult New Zealand rabbits, injected subcutaneously with two injections of 10 to 20 mug of norethindrone, 24 hr apart, and challenged intradermally with S. aureus 4 hr after the second injection, had fewer lesions with smaller areas of swelling and erythema as compared to control, nontreated rabbits. The protective effect of norethindrone on the development of staphylococcal lesion seemed related to hormone concentration. Thus, it was demonstrated with doses of 20, 15, and 10 mug, but not with doses of 1 and 5 mug. When the lesions were excised 48 to 92 hr after infection and when viable cell counts were made, rabbits treated with norethindrone showed significantly lower staphylococcal counts than the control rabbits. During the 1st day after infection with S. aureus, leukocytic counts of the norethindrone-treated rabbits remained normal, whereas control animals showed elevated leukocytic counts.     

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 Thymidine Labelling In Human and Porcine Growth Plates

Abstract. An in vitro technique has been used to label dividing cells in the growth plates of human bones with tritiated thymidine. the patterns of labelling in autoradiographs of human plates are described and values given for the labelling index, the number of cells in the proliferation zones and the heights of hypertrophied cells. In two of the four subjects no labelled cells were found in the growth plates and possible causes for these failures are discussed. In vitro labelling data on five porcine growth plates are also presented for comparison with the human data. In both structure and cell kinetics the epiphyseal cartilage plate in the pig is intermediate between the human and rodent plates.     

Cleavage of Viral Precursor Proteins In Vivo and In Vitro

The use of protease inhibitors causes the accumulation of very large polypeptides (polyprotein) in tissue culture cells infected with either poliovirus or echovirus 12. The effectiveness of the inhibitor varies, depending on the cell line chosen. In infected monkey kidney cells, polyprotein is not cleaved when a chymotrypsin inhibitor is added, but in infected HeLa cells a trypsin inhibitor is most effective. Therefore, at least a part of the proteolytic activity is supplied by the host cell. Extracted viral polyprotein can be cleaved in vitro by trypsin or chymotrypsin. As estimated by migration in sodium dodecyl sulfate gels and antigenicity, chymotrypsin cleavage of the poliovirus polyprotein yields fragments which are similar to the in vivo product. The polyprotein is not in soluble form but is attached to a fast-sedimenting, membrane-bound structure. Proteolytic activities in cell extracts were assayed using polyprotein as substrate, and infected and uninfected extracts produced qualitatively dissimilar cleavages.     

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.