Buffer gas cooled ice chemistry. II. Ice generation and mm-wave detection of molecules desorbed from an ice.

This second paper in a series of two describes the chirped-pulse ice apparatus that permits the detection of buffer gas cooled molecules desorbed from an energetically processed ice using broadband mm-wave rotational spectroscopy. Here, we detail the lower ice stage developed to generate ices at 4 K, which can then undergo energetic processing via UV/VUV photons or high-energy electrons and which ultimately enter the gas phase via temperature-programmed desorption (TPD). Over the course of TPD, the lower ice stage is interfaced with a buffer gas cooling cell that allows for sensitive detection via chirped-pulse rotational spectroscopy in the 60-90 GHz regime. In addition to a detailed description of the ice component of this apparatus, we show proof-of-principle experiments demonstrating the detection of H2CO products formed through irradiation of neat methanol ices or 1:1 CO + CH4 mixed ices.

Activation of the cannabinoid receptor 2 increases renal perfusion.

Acute kidney injury (AKI) is an increasing clinical problem that is associated with chronic kidney disease progression. Cannabinoid receptor 2 (CB2) activation has been shown to mitigate some of the deleterious tubular effects due to AKI, but its role on the renal vasculature has not been fully described. In this study, we investigated the effects of our novel CB2 receptor agonist, SMM-295, on renal vasculature by assessing cortical perfusion with laser Doppler flowmetry and changes in luminal diameter with isolated afferent arterioles. In this study, intravenously infused SMM-295 (6 mg/kg) significantly increased cortical renal perfusion (13.8 ± 0.6%; P < 0.0001; n = 7) compared with vehicle (0.1 ± 1.5%; n = 10) normalized to baseline values in anesthetized C57BL/6J mice. This effect was not dependent upon activation of the CB1 receptor (met-anandamide; 6 mg/kg iv) and was predominantly abolished in Cnr2 knockout mice with SMM-295 (6 mg/kg iv). Ablation of the renal afferent nerves with capsaicin blocked the SMM-295-dependent increase in renal cortical perfusion, and the increased renal blood flow was not dependent upon products synthesized by cyclooxygenase or nitric oxide synthase. The increased renal perfusion by CB2 receptor activation is also attributed to a direct vascular effect, since SMM-295 (5 µM) engendered a significant 37 ± 7% increase ( P < 0.0001; n = 4) in luminal diameters of norepinephrine-preconstricted afferent arterioles. These data provide new insight into the potential benefit of SMM-295 by activating vascular and nonvascular CB2 receptors to promote renal vasodilation, and provide a new therapeutic target to treat renal injuries that impact renal blood flow dynamics.

Homology modeling using multiple molecular dynamics simulations and docking studies of the human androgen receptor ligand binding domain bound to testosterone and nonsteroidal ligands.

To facilitate the rational design of novel and more potent androgen receptor ligands, three-dimensional models for the human androgen receptor ligand binding domain bound to testosterone have been developed. These models of the androgen receptor were based on the crystal structure of the highly homologous human progesterone receptor ligand binding domain. The homology modeled androgen receptor was refined using unrestrained multiple molecular dynamics simulations in explicit solvent. Key H-bonding partners with the 17-hydroxy group and 3-keto group of testosterone are Asn705 and Thr877, and Gln711 and Arg752, respectively. These models show the presence of a unique unoccupied cavity within the androgen receptor binding pocket which may be valuable in the development of novel selective androgen receptor ligands. A qualitative analysis of amino acid mutations within the hAR binding pocket that affect ligand binding are consistent with these androgen receptor models. In addition to testosterone, the binding modes of several hydroxyflutamide-like nonsteroidal ligands for the androgen receptor are investigated using flexible docking with FlexX followed by refinement of the initial complexes with molecular dynamics simulations. These docking studies indicate that Asn705 is an important determinant in binding hydroxyflutamide and its derivatives by participating in H-bond interactions with the alpha-hydroxy moiety of these ligands. In addition, the nitro functionality mimics the 3-keto group of the natural ligand testosterone and is involved in H-bonding interactions with Gln711 and Arg752. From these docking studies, we suggest a mechanism for the enantioselective binding of chiral hydroxyflutamide derivatives and expand upon the previously reported structure-activity relationship for hydroxyflutamide and its derivatives.

Primary virus isolation by a satellite laboratory.

A hospital-based satellite laboratory designed to isolate viral pathogens frequently found in renal transplant and hematology-oncology patients was compared with a system based on isolation by a state-wide reference laboratory. The hospital-based laboratory identified 12 viral isolates from the total 97 clinical specimens submitted. The hospital-based laboratory identified seven viral isolates from the first 50 clinical specimens, whereas the statewide reference laboratory identified six isolates from identical paired specimens. The average time from specimen collection to reporting results for the 97 specimens was 9.2 days for the hospital laboratory, as compared with 11.2 days for the first 50 duplicate specimens sent to the state laboratory. The cost per clinical specimen was $18.10, which compares favorably with costs for routine aerobic bacteriology. The use of a satellite laboratory system for primary viral isolation appears to provide rapid, accurate, and accessible viral diagnosis at an affordable cost.

Endocannabinoid crosstalk between placenta and maternal fat in a baboon model (Papio spp.) of obesity.

INTRODUCTION: Maternal obesity (MO) remains a serious obstetric problem with acute and chronic morbidities for both mothers and offspring. The mechanisms underlying these adverse consequences of MO remain unknown. Endocannabinoids (ECB) are neuromodulatory lipids released from adipocytes and other tissues. Metabolic crosstalk between placenta and adipocytes may mediate sequelae of MO. The goal of this study was to elucidate placental and systemic ECB in MO. MATERIAL AND METHODS: Placentas, sera, and subcutaneous fat were collected at Cesarean sections performed near term (0.9 G) in four non-obese (nOB) and four obese (OB) baboons (Papio spp.). Concentrations of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were measured by liquid chromatography coupled to tandem mass spectrometry. AEA and 2-AG pathways were characterized in placentas by Q-RT-PCR, Western blot and immunohistochemistry. RESULTS: Placental 2-AG levels were lower and maternal fat AEA levels were higher in OB (1254.1 ± 401.3 nmol/kg and 17.3 ± 4 nmol/kg) vs. nOB (3124.2 ± 557.3 nmol/kg and 3.1 ± 0.6 nmol/kg) animals. Concentrations of 2-AG correlated positively between maternal fat and placenta (r = 0.82, p = 0.013), but correlated negatively with maternal leptin concentrations (r = -0.72, p = 0.04 and r = -0.83, p = 0.01, respectively). CONCLUSION: This is the first study to demonstrate differential ECB pathway regulation in maternal fat and placenta in MO. Differential regulation and function exist for AEA and 2-AG as the major ECB pathways in placenta.

Utilization of health care services by pregnant mothers during delivery: a community based study in Nigeria.

OBJECTIVE: Poor utilization of health facilities during delivery by pregnant mothers is still a major cause of maternal and childhood morbidity and mortality in Nigeria. The aim of this study was to determine the level of utilization of health care services by pregnant women during delivery in Gokana Local Government Area of River State, Nigeria. MATERIALS AND METHODS: This was a cross-sectional, questionnaire; based study involving 112 mothers aged 15 years to 49 years from Gokana Local Government Area of Rivers State, Nigeria. The local Government Area has 12 health centres and 6 health centres were selected by multistage sampling. 112 were then selected by simple random sampling. RESULTS: Of the 112 mothers interviewed 91 (81.3%) were married, 13 (11.6%) were single, 5 (4.5%) were widows, 2 (1.8%) divorced and 1 (0.9%) separated. Ninety seven (86.6%) of these mothers (n = 112) had formal education while 15 (13.4%) had no formal education. Most 42 (37.5%) of the mothers were between 25-29 years. Sixty four (57.1%) of the 112 mothers in their recent delivery used a health facility while 48 (42.9%) did not. Factors responsible for non utilization of health facility for delivery include: Long distance to health facility 33 (68.7%), onset of labour at night 40 (83.3%), unavailability of means of transportation 37 (77.1%), Lack of money for transportation 26 (54.2%), unsatisfactory services at health facility 26 (54.2%), unfriendly attitude of staff of the health facility 34 (70.8%), unavailability of staff at health facility 32 (64.0%), lack of urgency at health facility 36 (75.0%), previous uneventful delivery at the health facility 32 (66.7%). CONCLUSION: Utilization of health care services during delivery in Nigeria is still poor. Concerted efforts should be made both at community and Government levels to improve utilization of health facility during delivery. This will go a long way in reducing maternal and child mortality.

Mini-chromatofocusing of plant and fungal polyphenoloxidases.

Mini-chromatofocusing was used to estimate the isoelectric points of mung bean polyphenoloxidase (5.4), broad bean polyphenoloxidase (5.4-5.7), and mushroom tyrosinase (4.5) in crude and purified samples. Using 1 to 2 ml of Polybuffer exchanger and 20 to 25 ml of Polybuffer 74, isoelectric points for the above enzymes were obtained in less than 2 h. Using this technique, isoelectric points for beta-lactoglobulin (4.5), carbonic anhydrase (6.4), soybean trypsin inhibitor (4.1), myoglobin (6.9), and bovine serum albumin (4.8) were obtained and compared to existing literature values. Mini-chromatofocusing provides a rapid estimation for isoelectric points of proteins and enzymes and may be a useful alternative to conventional methods for determination of isoelectric points.

Sodium dodecyl sulfate activation of a plant polyphenoloxidase. Effect of sodium dodecyl sulfate on enzymatic and physical characteristics of purified broad bean polyphenoloxidase.

Latent broad bean polyphenoloxidase was purified and shown to be activated by sodium dodecyl sulfate (SDS). Further characterization of the enzyme was carried out in the presence and absence of SDS. Activation of the enzyme increased in a sigmoidal manner with increasing SDS concentrations up to a maximum of 1.75 mM. The presence of SDS eliminated a low pH optimum induced by acid shocking. Increased thermolability of the enzyme was observed in the presence of SDS as well as an increased binding of [14C]dihydroxy-phenylalanine. Size exclusion chromatography on high performance liquid chromatography showed that the size and apparent molecular mass of the enzyme were slightly altered in the presence (48 kDa) versus absence (47 kDa) of SDS. Although the estimations were larger than those obtained by size exclusion techniques, no large differences in molecular weight were observed after sedimentation equilibrium of the enzyme in the presence (53.9 kDa) and absence (52.3 kDa) of SDS. Relative electrophoretic mobility and intrinsic fluorescence of tyrosine and tryptophan residues increased in a complex fashion as the SDS concentration was increased. Plateau regions in these latter experiments corresponded to concentrations of SDS needed for activation. The ability of SDS to activate the enzyme alters both its enzymatic and physical characteristics and suggests that a limited conformational change, due to binding of small amounts of SDS, may induce or initiate the activation of latent enzyme.

Histochemical localization of mushroom tyrosinase in whole tissue sections on nitrocellulose.

Mushrooms were cut into vertical and horizontal sections. These sections were blotted onto nitrocellulose sheets and the sheets were then stained for tyrosinase using L-dopa. Tyrosinase was localized throughout the mushroom tissues but more enzyme was located in the epidermis of the cap, the gill region, and the stipe. Preincubation of the nitrocellulose sheets in specific inhibitors of tyrosinase completely blocked enzyme staining, suggesting that the enzyme stained areas on the nitrocellulose blots were regions of tyrosinase activity. Immunochemical localization of tyrosinase was similar to that observed by histochemical staining. Nitrocellulose blotting of mushrooms allows localizations of enzyme at the whole tissue level and may be useful for other enzymes in mushrooms as well.

Light-scattering effects in the measurement of membrane microviscosity with diphenylhexatriene.

Data from several membrane systems are presented to confirm an empirical means of correcting diphenylhexatriene fluorescence for depolarization caused by sample turbidity. The depolarization proportionally constants obtained are not equal, but are shown to vary with (a) the physical state of the membrane, (b) the cholesterol content of the membrane, (c) the protein content of the membrane, and (d) the method of membrane preparation or isolation. It is concluded that depolarization corrections must always be considered when diphenylhexatriene fluorescence anisotropy is used to compare the fluidities within different membrane bilayers.

Deoxyadenosine-based DNA polymerase photoprobes: design, synthesis, and characterization as inhibitors of the Escherichia coli DNA polymerase I Klenow fragment.

DNA polymerase photoprobes 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate (1), 2-[(4-azidophenylsulfenyl)thio]-2'-deoxyadenosine 5'-triphosphate (2), and 2-[(4-azido-2-nitrophenyl)-thio]-2'-deoxyadenosine 5'-triphosphate (3) were designed from a thermodynamic model of DNA polymerase 1-substrate interactions such that the triphosphate would anchor the inhibitor and allow the phenyl azide to interact with the complementary template binding site. Photoprobes 1-3 were synthesized by condensation of 2-thio-2'-deoxyadenosine or its phosphate with p-azidophenacyl bromide, N-(4-azidophenylsulfenyl)phthalimide, and 4-azido-1-fluoro-2-nitrobenzene, respectively, and characterized as reversible and photoinduced irreversible inhibitors of the DNA polymerase I Klenow fragment and HIV I reverse transcriptase. The aryl azides decomposed with irradiation at 300 and 350 nm with half-lives ranging from 0.98 to 2.33 min and 2.15 to 5.38 min, respectively, with quantum efficiencies ranging from 0.29 to 0.55 and no apparent photodecomposition of the 2-thio-2'-deoxyadenosine nucleotide. Photoprobes 1-3 showed mixed noncompetitive inhibition of the Klenow fragment polymerase activity versus poly(dA).(T)10 as variable substrate with apparent competitive inhibition constants of 2.1, 36, and 29 microM, respectively, evidence suggesting that these photoprobes bind to both the free enzyme form and the enzyme-template-primer binary complex. Of the three photoprobes, only nucleotide 1 photoinactivates the Klenow fragment; in the presence of a 200-fold excess of nitrene scavenger, photoprobe 1 inactivates 92% of the Klenow fragment polymerase activity with saturation observed at 9.7 microM and an IC50 of about 2 microM. This evidence demonstrates that photoprobe 1 does bind to the Klenow fragment in the absence of template-primer and that it is an efficient photoprobe.

DNA polymerase photoprobe 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate labels an Escherichia coli DNA polymerase I Klenow fragment substrate binding site.

The nucleotide photoprobe 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate (1) was evaluated as a photoaffinity label of the DNA polymerase I Klenow fragment. Photolabel [3H]-1 covalently labeled the Klenow fragment with photolysis at 300 nm, reaching saturation at an approximate 1:1 mole ratio at 5.7 microM and with an EC50 (the effective concentration at 50% maximum photoincorporation) of about 0.74 microM. Saturating concentrations of poly(dA).(T)10 protect the Klenow fragment from [3H]-1 photoincorporation, and TTP at a concentration approximately equal to its KD for the free enzyme form shifts the dose-response curve for photoincorporation of [3H]-1 into the Klenow fragment by a factor of 2, indicating a competitive relationship between TTP and 1. Additionally, the photoincorporation of [3H]-1 into the Klenow fragment has an absolute requirement for magnesium, with no significant photoincorporation observed at concentrations of 1 up to 10 microM in the absence of magnesium. These results demonstrate that, as designed, photoprobe 1 binds to both the dNTP and a portion of the template-primer binding sites on the Klenow fragment. Photoaffinity labeling of the Klenow fragment by 1 yielded a single radiolabeled tryptic fragment which was isolated by HPLC; sequence analysis identified Asp732 in the peptide fragment Asp732-Ile733-His734-Arg735 as the site of covalent modification. Molecular modeling and complementary NMR analysis of the conformation of 1 indicated preferred C3'-exo and C2'-exo-C3'-endo symmetrical twist furanose ring puckers, with a high antibase conformation and a +sc C-5 torsional angle. Docking studies using Asp732 as an anchor point for the azide alpha-nitrogen on the photolabel indicate that the dNTP binding site is at the edge of the DNA binding cleft opposite the exonuclease site and that the template binding site includes helix O in the finger motif of the Klenow fragment.

Effect of lipid membrane structure on the adenosine 5'-triphosphate hydrolyzing activity of the calcium-stimulated adenosinetriphosphatase of sarcoplasmic reticulum.

An active Ca2+-stimulated, Mg2+-dependent adenosinetriphosphatase (Ca2+-ATPase) isolated from rabbit skeletal muscle sarcoplasmic reticulum membranes has been incorporated into dilauroyl-, dimyristoyl-, dipentadecanoyl-, dipalmitoyl-, and palmitoyloleoylphosphatidylcholine bilayers by using a newly developed lipid-substitution procedure that replaces greater than 99% of the endogenous lipid. Freeze--fracture electron microscopy showed membranous vesicles of homogeneous size with symmetrically disposed fracture-face particles. Diphenylhexatriene fluorescence anisotropy was used to define the recombinant membrane phase behavior and revealed more than one transition in the membranes. Enzymatic analysis indicated that saturated phospholipid acyl chains inhibited both overall ATPase activity and Ca2+-dependent phosphoenzyme formation below the main lipid phase transition temperature (Tm) of the lipid-replaced membranes. At temperatures above Tm, ATPase activity but not phosphoenzyme formation was critically dependent on acyl chain length and thus bilayer thickness. No ATPase activity was observed in dilauroylphosphatidylcholine bilayers. Use of the nonionic detergent dodecyloctaoxyethylene glycol monoether demonstrated that the absence of activity was not due to irreversible inactivation of the enzyme. Increased bilayer thickness resulted in increased levels of activity. An additional 2-fold rise in activity was observed when one of the saturated fatty acids in dipalmitoylphosphatidylcholine was replaced by oleic acid, whose acyl chain has a fully extended length comparable to that of palmitic acid. These results indicate that the Ca2+-ATPase requires for optimal function a "fluid" membrane with a minimal bilayer thickness and containing unsaturated phospholipid acyl chains.

Evaluation of three newer methods for investigating protein interactions of penicillin G.

THE INTERACTION OF PENICILLIN G WITH HUMAN SERUM PROTEINS WAS EVALUATED BY THREE DIFFERENT TECHNIQUES: rate of dialysis, cross-linked dextran exclusion, and ultracentrifugation. The rate-of-dialysis technique demonstrated that penicillin G binding to serum was immediate but incompletely reversible. Cross-linked dextran adsorbed or trapped significant amounts of penicillin G, necessitating correction factors of more than 10%. Ultracentrifugation was found to be the most reliable method for quantitative protein-binding determinations of penicillins.

Effects of age, race, sex, and smoking on prothrombin fragment 1.2 in a healthy population.

Prothrombin fragment 1.2 (F1.2) is a biomarker of thrombin generation during blood coagulation and has diagnostic potential for assessing thrombotic risk and monitoring anticoagulation therapy. We used a monoclonal antibody-based immunoassay for plasma F1.2 to establish a well-defined reference interval and to evaluate the effects of age, race, sex, and smoking status on F1.2 concentrations in a healthy population. Plasma samples and demographic information were obtained from 357 healthy individuals. F1.2 concentrations more closely followed a lognormal than a gaussian frequency distribution. In a multiple linear-regression model in which the logarithms of F1.2 concentrations were regressed on age, race, sex, and smoking status, the significant explanatory variables were age and, to a lesser extent, sex and smoking. A segmented "hockey stick" regression model indicated that F1.2 concentrations and age were unrelated for individuals < 44 years old but were positively correlated above that age threshold. The estimated 95% tolerance interval (P = 0.95) for F1.2 in healthy individuals < 44 years old (n = 268) was 0.21-2.78 nmol/L. We conclude that age-matched F1.2 reference intervals may be important for studies evaluating the diagnostic utility of F1.2 measurements, and that the clinical relevance of increased thrombin generation during aging warrants further investigation.

Evaluation of preanalytical variables associated with measurement of prothrombin fragment 1.2.

We have used a monoclonal antibody-based ELISA for plasma prothrombin fragment 1.2 (F1.2) to establish appropriate sample collection and storage conditions for this biomarker of thrombin generation. F1.2 concentrations were not altered by exogenous factor Xa, thrombin, or thromboplastin if blood was collected by routine venipuncture into tubes containing heparin as anticoagulant (but not citrate, acid-citrate-dextrose, EDTA, or oxalate) and if plasma antithrombin III concentration was > or = 30% of normal. Heparinized plasma F1.2 was stable for > or = 8 h at 20-25 degrees C, and if premixed with a stabilizing reagent, for > or = 4 years at -70 degrees C. Mean values for heparinized plasma F1.2 collected and stored by recommended procedures were increased in patients with thrombosis and conditions of increased thrombotic risk, and were sensitive to heparin and oral anticoagulant therapies. We conclude that plasma obtained by routine venipuncture into tubes with heparin as anticoagulant is an appropriate specimen for F1.2 measurements for most patients.

Survival of influenza viruses on environmental surfaces.

To investigate the transmission of influenza viruses via hands and environmental surfaces, the survival of laboratory-grown influenza A and influenza B viruses on various surfaces was studied. Both influenza A and B viruses survived for 24-48 hr on hard, nonporous surfaces such as stainless steel and plastic but survived for less than 8-12 hr on cloth, paper, and tissues. Measurable quantities of influenza A virus were transferred from stainless steel surfaces to hands for 24 hr and from tissues to hands for up to 15 min. Virus survived on hands for up to 5 min after transfer from the environmental surfaces. These observations suggest that the transmission of virus from donors who are shedding large amounts could occur for 2-8 hr via stainless steel surfaces and for a few minutes via paper tissues. Thus, under conditions of heavy environmental contamination, the transmission of influenza virus via fomites may be possible.