Synthesis of aromatic nitrogen mustard agents and analysis of their alkylation activity at physiological pH and temperature

A Structure Activity Relationship (SAR) study was accomplished with six aromatic compounds which have a nitrogen mustard (N-mustard) substituent. N-mustard agents are very important for the clinical treatment of many types of cancers. All N-mustard agents synthesized alkylated a nucleophilic primary amine (p-chloroaniline) in aqueous solvent at pH 7.4 and 37 degrees C. Rate constants and rate equations were determined for the alkylation reactions by monitoring the formation of a fluorescent complex formed when fluorescamine complexes the unreacted p-chloroaniline. Fluorescamine complexation of the unreacted primary amine halts the alkylation reaction and allows the determination of remaining unreacted primary amine, which in turn permits the determination of rate constants and rate equations. The fluorescamine-amine complex shows a strong absorbance peak at a wavelength of 400 nanometers in aqueous solvent. The molar absorptivity (epsilon) was calculated to be 18.37 L/(mole x cm). First order rate constants ranged from 0.513E-2 minute(-1) to 1.32E-2 minute(-1) with second order rate constants from 2.85E-2 (M x minute)(-1) to 4.78E-2 (M x minute)(-1). The aromatic compounds included benzoic acid, m-chlorobenzoic acid, hydrocinnamic acid, m-toluic acid, and 3,4-dimethoxybenzoic acid. The synthesis procedure produced the N-mustard agents at > or = 90% yield and high purity. Partition coefficient Log(Kow)Log P values ranged from 2.66 to 4.18. The N-mustard agents consisted of greyish-yellow crystals which retained alkylation activity for more than ten weeks when stored at -10 degrees C and were soluble in water at 25 degrees C and 37 degrees C.     

Molecular properties and antibacterial activity of the methyl and ethyl ester derivatives of ampicillin

Ampicillin is a beta-lactam antibiotic that is effective against gram-negative bacteria. Ampicillin has a single carboxyl group (-C(O)OH) within its structure which is suitable for forming ester compounds. Diazomethane and diazoethane were utilized to react with ampicillin to form the methyl and ethyl esters, respectively. The ester derivatives of ampicillin were solubilized together (mole ratio 1:1) in LB media and penicillin resistant Escherichia coli added to measure antibacterial activity. Growth inhibition of bacteria was monitored by optical density after a known time period and with known specific concentrations of the ampicillin esters present. Significant growth inhibition of penicillin resistant bacteria occurred at concentrations of the combined methyl and ethyl ampicillin esters from less than 50 microgram/mL to more than 150 microgram/mL. Molecular properties of the ester compounds were determined. The two ester derivatives showed values of Log BB, Log P, polar surface area, intestinal absorption, and solubility suitable for clinical application. The two ester compounds showed zero violations of the Rule of 5 indicating good bioavailability. The two ester derivatives showed greater intestinal absorbance and greater penetration of the blood brain barrier than the parent ampicillin. Favorable druglikeness was determined for both ester derivatives.     

Assessment of penetration of quantum dots through in vitro and in vivo human skin using the human skin equivalent model and the tape stripping method

Quantum dots (QDs) are rapidly emerging as an important class of nanoparticles (NPs) with potential applications in medicine. However, little is known about penetration of QDs through human skin. This study investigated skin penetration of QDs in both in vivo and in vitro human skin. Using the tape stripping method, this study demonstrates for the first time that QDs can actually penetrate through the stratum corneum (SC) of human skin. Transmission electron microscope (TEM) and energy diverse X-ray (EDX) analysis showed accumulation of QDs in the SC of a human skin equivalent model (HSEM) after dermal exposure to QDs. These findings suggest possible transdermal absorption of QDs after dermal exposure over a relatively long period of time.     

Expression and role of E- and P-cadherin adhesion molecules in embryonic histogenesis. II. Skin morphogenesis

Expression and the role of E- and P-cadherin in the histogenesis of the surface epidermis and hair follicles were examined using the upper lip skin of the mouse. P-cadherin is expressed exclusively in the proliferating region of these tissues, that is in the germinative layer of the surface epidermis, the outer root sheath and the hair matrix. E-cadherin is coexpressed in these layers but this molecule was also detected in non-proliferating regions such as the intermediate layer of the surface epidermis and the immature regions of the inner root sheath. Neither P- nor E-cadherin was detected in fully keratinized layers such as the horny layer of the surface epidermis, the outermost layer of the outer root sheath and the mature hair fibres. These two cadherins were not detected in dermal cells. We cultured pieces of the upper lip skin in vitro in the absence or presence of a monoclonal antibody to E-cadherin (ECCD-1) or to P-cadherin (PCD-1). In control cultures, skin morphogenesis normally occurred in a pattern whereby the hair follicles grew and dermal cells were condensed to form the dermal sheath. A mixture of ECCD-1 and PCD-1, however, induced abnormal morphogenesis in the skin in several respects. (1) The cuboidal or columnar arrangement of basal epithelial cells was distorted. (2) Hair follicles were deformed. (3) Condensation of dermal cells was suppressed, causing a homogeneous distribution of these cells. These results suggest that cadherins present in epidermal cells are involved not only in maintaining the arrangement of these cells but also in inducing dermal condensation.     

Schistosoma mansoni: correlations between mouse strain, skin eicosanoid production, and cercarial skin penetration

We have previously reported that cercarial penetration is highly correlated with cercarial production of leukotrienes (LT's) and hydroxyeicosatetraenoic acids (HETE's). Because skin also produces various eicosanoids, we undertook an investigation of skin eicosanoids in various strains of mice and 1 strain of rat in order to ascertain if skin eicosanoids could be correlated to cercarial penetration. SENCAR, ICR, NMRI, A/J, C3H/HeJ, C57Bl/6, ASEBIC, and BALB/c mouse strains were used in this study as well as the SD-Rat strain. The ability of cercariae to penetrate skin was strain specific. A/J and SENCAR mice had the highest penetration rates (approximately 98%), whereas the SD-Rat strain had the lowest (43%). These penetration rates we linearly correlated with tail skin HETE production at 10 min (R = 0.826), whereas HETE production at 60 min had a parabola-shaped relationship (R = 0.793). The primary infection of mice with Schistosoma mansoni cercariae may therefore be directly correlated with both the skin's innate ability to synthesize HETE, as well as with cercarial eicosanoid production, especially HETE levels. However, we believe that skin eicosanoid production is just one of many factors affecting cercarial skin penetration. Other factors discussed are: skin surface fatty acid levels, cercarial eicosanoid production, epidermal vs. dermal eicosanoid production, and the immunocompetence of the host.     

大肠埃希菌和肺炎克雷伯菌ESBLs检测及耐药性分析

由细菌超广谱β-内酰胺酶（ESBLs）引起的细菌耐药性一直是临床相关感染性疾病治疗中的棘手问题。从不同病区患者标本中分离了96株大肠埃希菌和80株肺炎克雷伯菌,分剐采用双纸片协同试验和药物敏感试验检测了上述菌株产生ESBLs情况及对17种抗生素的耐药性。结果发现,27．1％（26／96）的大肠埃希菌株和22．5％（18／80）肺炎克雷伯菌株产ESBLs。ICU病房分离的大肠埃希菌和肺炎克雷伯菌株ESBLs总阳性率（46．0％）与介入科病房和烧伤科病房分离菌株ESBLs总阳性率（28．6％和25．0％）无显著性差异（P〉0．05）,但明显高于呼吸科、骨科、其他病房及门诊部分离菌株ESBLs总阳性率（6．3％～14．3％,P〈0．01）。不产ESBLs大肠埃希菌株和肺炎克雷伯菌株对17种抗生素耐药率明显低于产ESBLs菌株。产ESBLs大肠埃希菌和肺炎克雷伯菌对氨曲南均敏感,对氨苄西林／舒巴坦、阿莫西林／棒酸、阿米卡星耐药率仅为15．8％-23．4％。上述实验结果提示,大肠埃希菌和肺炎克雷伯菌临床菌株中有较高的ESBLs阳性率,不同病区患者感染的大肠埃希菌和肺炎克雷伯菌ESBLs阳性率有很大差异,氨曲南、氨苄西林／舒巴坦、阿莫西林／棒酸、阿米卡星可作为治疗产ESBLs大肠埃希菌和肺炎克雷伯菌感染性疾病的首选药物。     

In Vitro Penetration of Pig Skin by Heavy Metals in Soil

The potential health risk from exposure to heavy metal contaminated soil is often based on the quantity of metal that can be removed from soil by vigorous extraction procedures. This approach can overestimate risk since it ignores complex interactions between metals and soil that can result in a reduction in the amount of metal that desorbs from soil and is subsequently absorbed by the body. The aim of this research was to determine the relative contribution of the soil matrix and heavy metal sequestration in soil with time (“aging”) on the dermal penetration of arsenic, mercury, and nickel, respectively, as arsenic acid, mercuric chloride, and nickel chloride. In vitro flow-through diffusion cell studies were performed utilizing dermatomed male pig skin and radioactive compounds to measure total penetration (the sum of each metal in receptor fluid and skin). For arsenic and nickel, the soil matrix produced a 78–87% reduction in dermal penetration compared to 12–19% after aging. A greater effect was observed with aged mercury (52–56% decrease in dermal penetration) than in freshly spiked soil (40–43%). The results indicate that the potential health risk from dermal exposure to the metals can be significantly reduced by soil and aging.     

Simulation of electron-beam irradiation of skin tissue model

Monte Carlo simulation of electrons stopping in liquid water was used to model the penetration and quality of electron-beam irradiation incident on the full-thickness EpiDerm? skin model (EpiDermFT? MatTek, Ashland, VA). This 3D tissue model has a fully developed basement membrane separating an epidermal layer of keratinocytes in various stages of differentiation from a dermal layer of fibroblasts embedded in collagen. The simulations were motivated by a desire to selectively expose the epidermal layer to low-linear energy transfer (LET) radiation in the presence of a nonirradiated dermal layer. The variable-energy electron microbeam at the Pacific Northwest National Laboratory (PNNL) was used as a model of device characteristics and irradiation geometry. At the highest beam energy available (90 keV), we estimate that no more than a few percent of the beam energy will be deposited in the dermal layer. Energy deposition spectra were calculated for 10-μm-thick layers near the 10th, 50th and 90th percentiles of penetration by the 90 keV electron beam. Bimodal spectra showed an increasing component of "stoppers" with increasing depth, which increases the probability of large energy deposition events. Nevertheless, screening by tissue above the layer of interest is the main factor determining energy deposited at a given depth.     

N-(4-bromobenzoyl)-S,S-dimethyliminosulfurane, a potent dermal penetration enhancer

N-Aroyl-, N-Arylsulfonyl-, and N-Aryl-S,S-dimethyliminosulfuranes have been synthesized and evaluated as potential dermal penetration enhancers. The title compound and Azone exhibit similar activities for permeation of hydrocortisone through hairless mouse skin.     

Cream Formulation Impact on Topical Administration of Engineered Colloidal Nanoparticles

In order to minimize the impact of systemic toxicity of drugs in the treatment of local acute and chronic inflammatory reactions, the achievement of reliable and efficient delivery of therapeutics in/through the skin is highly recommended. While the use of nanoparticles is now an established practice for drug intravenous targeted delivery, their transdermal penetration is still poorly understood and this important administration route remains almost unexplored. In the present study, we have synthesized magnetic (iron oxide) nanoparticles (MNP) coated with an amphiphilic polymer, developed a water-in-oil emulsion formulation for their topical administration and compared the skin penetration routes with the same nanoparticles deposited as a colloidal suspension. Transmission and scanning electron microscopies provided ultrastructural evidence that the amphiphilic nanoparticles (PMNP) cream formulation allowed the efficient penetration through all the skin layers with a controllable kinetics compared to suspension formulation. In addition to the preferential follicular pathway, also the intracellular and intercellular routes were involved. PMNP that crossed all skin layers were quantified by inductively coupled plasma mass spectrometry. The obtained data suggests that combining PMNP amphiphilic character with cream formulation improves the intradermal penetration of nanoparticles. While PMNP administration in living mice via aqueous suspension resulted in preferential nanoparticle capture by phagocytes and migration to draining lymph nodes, cream formulation favored uptake by all the analyzed dermis cell types, including hematopoietic and non-hematopoietic. Unlike aqueous suspension, cream formulation also favored the maintenance of nanoparticles in the dermal architecture avoiding their dispersion and migration to draining lymph nodes via afferent lymphatics.     

Changes in antibiotic sensitivity in strains of Staphylococcus aureus, 1952-78.

Two hundred strains of Staphylococcus aureus isolated from outpatients with infections of the skin and subcutaneous tissues were tested for sensitivity to penicillin, erythromycin, tetracycline, sodium fusidate, methicillin, clindamycin, chloramphenicol, and gentamicin. One hundred and sixty-three (81.5%) of the strains were resistant to penicillin and 16 (8%) resistant to tetracycline. Incidence of resistance to other antibiotics was low. No strain was resistant to chloramphenicol, gentamicin, or methicillin. When compared with results of earlier studies, there was an increase in the incidence of resistance to penicillin and tetracycline, but no appreciable increase in resistance to other antibiotics.     

Murein and the Outer Penetration Barrier of Escherichia coli K-12, Proteus mirabilis, and Pseudomonas aeruginosa

A penetration barrier operating outside the periplasmic enzyme penicillinase was studied in an ampicillin-resistant mutant of Escherichia coli K-12. Growth in the presence of lysozyme and sublethal concentrations of ampicillin partially opened the barrier. This could be recorded as an increased penetration of penicillin G, sodium cholate, and rifampin to their respective targets. Brief treatments with tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid and sodium cholate effectively impaired the barrier against penicillin and also caused leakage of penicillinase. Wild-type E. coli K-12, Proteus mirabilis, and Pseudomonas aeruginosa also showed an increased sensitivity to cholate after treatment with penicillins. Electron micrographs showed that lysis by cholate was due to a distortion of the cytoplasmic membrane causing a leakage of protein and RNA from the cells to the medium. Physiological data indicated that the increased sensitivity to cholate induced by growth in the presence of ampicillin or lysozyme was due to effects upon the murein. This was supported by measurement of the incorporation of ³H-diaminopimelic acid. These results indicate that the murein sacculus either is a part of the penetration barrier or is responsible for holding the structure of the outer membrane together.     

Statistical modeling of ultrastructural features of murine dermal collagen under chronic low-dose whole body X-irradiation

The present study investigated the changes in ultrastructural features of dermal collagen fibrils of mice following exposure to different cumulative chronic low-dose X-irradiation through digital image analysis-based statistical modeling. Pubertal mice were X-irradiated and dorsal skin biopsies were collected and processed for transmission electron microscopic (TEM) analysis. TEM features of collagen fibrils showed alteration in the cross-sectional area, population density and in the axial periodic pattern of light and dark bands. The mathematical analysis of histogram data from TEM images revealed some adaptive behavior in collagen structures of the X-irradiated group. This finding indicated that exposure to chronic low-dose X-radiation induced an altered steady state with adaptive variation in dermal collagen fibrils in irradiated mice.     

In vivo percutaneous absorption of boron as boric acid, borax, and disodium octaborate tetrahydrate in humans: a summary

Literature from the first half of this century reports concern for toxicity from topical use of boric acid, but assessment of percutaneous absorption has been impaired by lack of analytical sensitivity. Analytical methods in this study included inductively coupled plasma-mass spectrometry, which now allows quantitation of percutaneous absorption of 10B in 10B-enriched boric acid, borax, and disodium octaborate tetrahydrate (DOT) in biological matrices. This made it possible, in the presence of comparatively large natural dietary boron intakes for the in vivo segment of this study, to quantify the boron passing through skin. Human volunteers were dosed with 10B-enriched boric acid, 5.0%, borax, 5.0%, or disodium octaborate tetrahydrate, 10% in aqueous solutions. Urinalysis, for boron and changes in boron isotope ratios, was used to measure absorption. Boric acid in vivo percutaneous absorption was 0.226 (SD = 0.125) mean percent dose, with flux and permeability constant (Kp) calculated at 0.009 microg/cm2/h and 1.9 x 10(-7) cm/h, respectively. Borax absorption was 0.210 (SD = 0.194) mean percent dose, with flux and Kp calculated at 0.009 microg/cm2/h and 1.8 x 10(-7) cm/h, respectively. DOT absorption was 0.122 (SD = 0.108) mean percent, with flux and Kp calculated at 0.01 microg/cm2/h and 1.0 x 10(-7) cm/h, respectively. Pretreatment with the potential skin irritant 2% sodium lauryl sulfate had no effect on boron skin absorption. These in vivo results show that percutaneous absorption of boron, as boric acid, borax, and disodium octaborate tetrahydrate, through intact human skin is low and is significantly less than the average daily dietary intake. This very low boron skin absorption makes it apparent that, for the borates tested, the use of gloves to prevent systemic uptake is unnecessary. These findings do not apply to abraded or otherwise damaged skin.     

Spectrally resolved multiphoton imaging of in vivo and excised mouse skin tissues

The deep tissue penetration and submicron spatial resolution of multiphoton microscopy and the high detection efficiency and nanometer spectral resolution of a spectrograph were utilized to record spectral images of the intrinsic emission of mouse skin tissues. Autofluorescence from both cellular and extracellular structures, second-harmonic signal from collagen, and a narrowband emission related to Raman scattering of collagen were detected. Visualization of the spectral images by wavelength-to-RGB color image conversion allowed us to identify and discriminate tissue structures such as epidermal keratinocytes, lipid-rich corneocytes, intercellular structures, hair follicles, collagen, elastin, and dermal cells. Our results also showed morphological and spectral differences between excised tissue section, thick excised tissue, and in vivo tissue samples of mouse skin. Results on collagen excitation at different wavelengths suggested that the origin of the narrowband emission was collagen Raman peaks. Moreover, the oscillating spectral dependency of the collagen second-harmonic intensity was experimentally studied. Overall, spectral imaging provided a wealth of information not easily obtainable with present conventional multiphoton imaging systems.     

Effects of human amniotic membrane grafts combined with marrow mesenchymal stem cells on healing of full-thickness skin defects in rabbits

We have investigated the wound-healing effects of mesenchymal stem cells (MSCs) in combination with human amniotic membrane (HAM) when grafted into full-thickness skin defects of rabbits. Five defects in each of four groups were respectively treated with HAM loaded with autologous MSCs (group A), HAM loaded with allologous MSCs (group B), HAM with injected autologous MSCs (group C), and HAM with injected allologous MSCs (group D). The size of the wounds was calculated for each group at 7, 12, and 15 days after grafting. The wounds were subsequently harvested at 25 days after grafting. Sections stained with hematoxylin and eosin were used to determine the quality of wound healing, as based on the characteristics and amount of granulated tissue in the epidermal and dermal layers. Groups A and B showed the most pronounced effect on wound closure, with statistically significant improvement in wound healing being seen on post-operative days 7, 12, and 15. Although a slight trend toward improved wound healing was seen in group A compared with group B, no statistically significant difference was found at any time point between the two groups. Histological examination of healed wounds from groups A and B showed a thin epidermis with mature differentiation and collagen bundle deposition plus recovered skin appendages in the dermal layer. In contrast, groups C and D showed thickened epidermis with immature epithelial cells and increased fibroblast proliferation with only partially recovered skin appendages in the dermal layer. Thus, the graft of HAM loaded with MSCs played an effective role during the healing of skin defects in rabbits, with no significant difference being observed in wound healing between autologous and allologous MSC transplantation. This study was supported by research funds from Dong-A University.     

Crystal structure of nicotinic acid mononucleotide adenylyltransferase from Pseudomonas aeruginosa in its Apo and substrate-complexed forms reveals a fully open conformation

The enzyme nicotinic acid mononucleotide adenylyltransferase (NaMN AT; EC 2.7.7.18) is essential for the synthesis of nicotinamide adenine dinucleotide and is a potential target for antibiotics. It catalyzes the transfer of an AMP moiety from ATP to nicotinic acid mononucleotide to form nicotinic acid adenine dinucleotide. In order to provide missing structural information on the substrate complexes of NaMN AT and to assist structure-based design of specific inhibitors for antibacterial discovery, we have determined the crystal structure of NaMN AT from Pseudomonas aeruginosa in three distinct states, i.e. the NaMN-bound form at 1.7A resolution and ATP-bound form at 2.0A as well as its apo-form at 2.0A. They represent crucial structural information necessary for better understanding of the substrate recognition and the catalytic mechanism. The substrate-unbound and substrate-complexed structures are all in the fully open conformation and there is little conformational change upon binding each of the substrates. Our structures indicate that a conformational change is necessary to bring the two substrates closer together for initiating the catalysis. We suggest that such a conformational change likely occurs only after both substrates are simultaneously bound in the active site.     

Determination of antimicrobial susceptibilities of clinically isolated anaerobic bacteria by E-test, ATB-ANA and agar dilution

A total of 60 anaerobic strains were isolated from 322 clinical specimens. These isolates were tested for susceptibility to seven antibiotics (penicillin G, amoxicillin/clavulanic acid, cefoxitin, imipenem, chloramphenicol, metronidazole, clindamycin) by using ATB-ANA and Epsilometer test (E-test) strips and the results were compared with the gold standard agar dilution method. Imipenem was found as the most effective agent in vitro among the agents tested (100%). Susceptibility to penicillin G, amoxicillin/clavulanic acid, cefoxitin, chloramphenicol, metronidazole and clindamycin are 36.7%, 83.3%, 88.3%, 96.6%, 85% and 90%, respectively. E-test has showed a good correlation (r=0.62, p=0.001) statistically with the results of agar dilution (total agreement for all antibiotics changing between 90.01% and 98.45%) and a moderate correlation (r=0.45, p=0.048) with the results of ATB-ANA method (total agreement for all antibiotics changing between 75.46% and 98.76%). However, the routine use of agar dilution procedure is concluded to be cumbersome, whereas E-test method offers a reliable alternative.     

Antibiotic penetration through the blood-brain barrier in rats and the effect on this process of proteolytic enzymes]

Penetration of penicillin, ampicillin, oxacillin, tetracycline, streptomycin and gentamycin through the hemato-encephalic barrier in rats and effect of proteolytic enzymes on the above process were studied comparatively. The levels of penetration to the brain were highest for ampicillin, then followed penicillin, tetracycline, oxacillin, streptomycin and gentamycin. Preliminary intramuscular administration of trypsin and chimotrypsin (10 mg/kg) to the animals had no effect on permeability of the hemato-encephalic barrier for the antibiotics tested, except penicillin. Higher levels of the antibiotics in the brain tissue may be explained by higher antibiotic blood levels under the effect of proteolytic enzymes.