Toward a new generation of therapeutics

Scientific evidence in the prevention and treatment of various disorders is accumulating regarding probiotics. The health benefits supported by adequate clinical data include increased resistance to infectious disease, decreased duration of diarrhea, management of inflammatory bowel disease, reduction of serum cholesterol, prevention of allergy, modulation of cytokine gene expression, and suppression of carcinogen production. Recent ventures in metabolic engineering and heterologous protein expression have enhanced the enzymatic and immunomodulatory effects of probiotics and, with time, may allow more active intervention among critical care patients. In addition, a number of approaches are currently being explored, including the physical and chemical protection of cells, to increase probiotic viability and its health benefits. Traditional immobilization of probiotics in gel matrices, most notably calcium alginate and κ-carrageenan, has frequently been employed, with noted improvements in viability during freezing and storage. Conflicting reports exist, however, on the protection offered by immobilization from harsh physiologic environments. An alternative approach, microencapsulation in “artificial cells,” builds on immobilization technologies by combining enhanced mechanical stability of the capsule membrane with improved mass transport, increased cell loading, and greater control of parameters. This review summarizes the current clinical status of probiotics, examines the promises and challenges of current immobilization technologies, and presents the concept of artificial cells for effective delivery of therapeutic bacterial cells.     

Oxidation of heme to beta- and delta-biliverdin by Pseudomonas aeruginosa heme oxygenase as a consequence of an unusual seating of the heme

The origin of the unusual regioselectivity of heme oxygenation, i.e. the oxidation of heme to delta-biliverdin (70%) and beta-biliverdin (30%), that is exhibited by heme oxygenase from Pseudomonas aeruginosa (pa-HO) has been studied by (1)H NMR, (13)C NMR, and resonance Raman spectroscopies. Whereas resonance Raman indicates that the heme-iron ligation in pa-HO is homologous to that observed in previously studied alpha-hydroxylating heme oxygenases, the NMR spectroscopic studies suggest that the heme in this enzyme is seated in a manner that is distinct from that observed for all other alpha-hydroxylating heme oxygenase enzymes for which a structure is known. In pa-HO, the heme is rotated in-plane approximately 110 degrees, so the delta-meso-carbon of the major orientational isomer is located within the HO-fold in the place where the alpha-hydroxylating enzymes typically place the alpha-meso-carbon. The unusual heme seating displayed by pa-HO places the heme propionates so that these groups point in the direction of the solvent-exposed heme edge and appears to originate in large part from the absence of stabilizing interactions between the polypeptide and the heme propionates, which are typically found in alpha-hydroxylating heme oxygenase enzymes. These interactions typically involve Lys-16 and Tyr-112, in Neisseriae meningitidis HO, and Lys-16 and Tyr-134, in human and rat HO-1. The corresponding residues in pa-HO are Asn-19 and Phe-117, respectively. In agreement with this hypothesis, we found that the Asn-19 Lys/Phe-117 Tyr double mutant of pa-HO exists as a mixture of molecules exhibiting two distinct heme seatings; one seating is identical to that exhibited by wild-type pa-HO, whereas the alternative seating is very similar to that typical of alpha-hydroxylating heme oxygenase enzymes and is related to the wild-type seating by approximately 110 degrees in-plane rotation of the heme. Furthermore, each of these heme seatings in the pa-HO double mutant gives rise to a subset of two heme isomeric orientations that are related to each other by 180 degrees rotation about the alpha-gamma-meso-axis. The coexistence of these molecules in solution, in the proportions suggested by the corresponding area under the peaks in the (1)H NMR spectrum, explains the unusual regioselectivity of heme oxygenation observed with the double mutant, which we found produces alpha- (55%), delta- (35%), and beta-biliverdin (10%). Alpha-biliverdin is obtained by oxidation of the heme seated similar to that of alpha-hydroxylating enzymes, whereas beta- and delta-biliverdin are formed from the oxidation of heme seated as in wild-type pa-HO.     

High throughput drug screening by direct RNA profiling on DNA sensors

The feasibility of DNA microarray sensor technology as a routine technique of molecular pharmacology to perform high throughput drug screening and the advantages of directly labeled RNA for a high throughput experiment are presented in this paper. A novel, single-step direct chemical labeling method for DNA microarray target samples has been developed to reduce the sample amount, cost, time and error of the experiment by eliminating the need for enzyme mediated labeling. Reproducibility of the data for high throughput drug screening is demonstrated by monitoring differential gene expression of a set of 45 gene targets involved in the genotoxic stress response pathways.     

Use of kinetic plots for relative assessment of reactor throughput and energy consumption

The progress of high temperature processes is generally described in terms of variation of the degree of conversion () with time (t). The present paper outlines a procedure for making use of-t plots for comparative assessment of productivity and energy requirements for a test system with respect to a reference, on the basis of some simplifying assumptions. It is assumed that the throughput is inversely proportional to reaction time as in the case of batch reactors and plug flow reactors. It is also assumed that the energy requirement is a simple function of process temperature. The principles outlined is illustrated with reference to some laboratory data for reduction of iron oxide by coal.The authors wish to thank Prof. P. R. Rao, Director National Metallurgical Laboratory, Jamshedpur, India, for providing facilities for experimental work and for according permission to publish this work.     

Efficiency of connected binary block designs when a single observation is unavailable

In this paper the problem of finding the design efficiency is considered when a single observation is unavailable in a connected binary block design. The explicit expression of efficiency is found for the resulting design when the original design is a balanced incomplete block design or a group divisible, singular or semiregular or regular with ₁>0, design. The efficiency does not depend on the position of the unavailable observation. For a regular group divisible design with ₁>0, the efficiency depends on the position of the unavailable observation. The bounds, both lower and upper, on the efficiency are given in this situation. The efficiencies of designs resulting from a balanced incomplete block design and a group divisible design are in fact high when a single observation is unavailable.The work of the first author is sponsored by the Air Force Office of Scientific Research under Grant AFOSR-90-0092.On leave from Indian Statistical Institute, Calcutta, India. The work of the third author was supported by a grant from the CMDS, Indian Institute of Management, Calcutta.     

Univariate Stochastic Programming with Random Decision Variable

The problem of minimising E(X) subject to the constraints X ? 0, P(X ? b) ? a(0 < a < 1) has been considered, where b is a non-negative random variable with continuous probability distribution. A necessary and sufficient condition for randomised decisions to be superior to the non-randomised one has been derived.     

A gamma-spectrometric method for the determination of plutonium isotope ratios

A gamma-spectrometric method using an intrinsic high resolution germanium detector has been developed for the determination of isotope ratios of plutonium from samples in solution form. The method is based on the assay of low energy gamma-rays of²³⁸Pu,²³⁹Pu,²⁴⁰Pu and²⁴¹Pu and does not require the use of branching intensities or the knowledge of detection efficiencies for different gamma rays. Since low energy gamma-rays are used, the effect of²⁴¹Am has also been studied. It is found that results are not affected up to 0.5 wt% of²⁴¹Am in plutonium samples. An accuracy of 3% is achievable in the determination of²⁴⁰Pu/²³⁹Pu and²⁴¹Pu/²³⁹Pu atom ratios as demonstrated by carrying out measurements on isotopic standards of plutonium.     

HPLC separation of hydrogenated derivatives of buckminsterfullerene

Summary The effect of composition and flow rate of the mobile phase on the HPLC separation of hydrogenated buckminsterfullerene (C₆₀H_n n=2–38) was investigated on BuckySep column. Toluene was used as the basic solvent and hexane, heptane, cyclohexane, THF, acetonitrile, acetone, ethanol and 2-propanol as co-solvents. The fraction of co-solvents was varied 10–80%, and the flow rate 1–0.1 mL min⁻¹. Toluene-acetonitrile 65∶35 and toluene-acetone 50∶50 provided the best separation. Under the best conditions complete separation of C₆₀H₂ and almost complete separation of the four most abundant isomers of C₆₀H₄ were achieved. Separation of derivatives with higher hydrogen content was very poor. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997     

The effects of gamma irradiation on the microbiological, physical and sensory qualities of diced tomatoes

Diced Roma tomatoes were treated with gamma irradiation and evaluated for changes in microbial, physical, chemical and sensory properties. Dosages for Trial 1 were 0.0, 0.39, 0.56 and 1.82 kGy and in Trial 2, 0.0, 0.50, 1.24 and 3.70 kGy. Irradiation at 3.70 kGy resulted in no aerobic populations through day 12 and significantly fewer colonies through day 15 whereas yeast and mold populations experienced a 2 log reduction through day 12. Color, titratable acidity, and °Brix were not significantly affected by irradiation. Tissue firmness decreased with increasing dose but not with storage time. Treatment with 3.7 kGy decreased firmness by 50% and 20% with 0.5 kGy, however, the reduced firmness induced by 0.50 kGy was undetected by a 9 member trained sensory panel. A significant (p0.05) inverse correlation between changes in texture and water-soluble pectin (WSP) was determined while total pectin remained relatively constant and oxalate soluble pectin content decreased slightly with irradiation dose. The significant inverse correlation between the loss of firmness and WSP indicates that the changes in WSP play an important role in the tissue softening of tomatoes, This study indicates that irradiation at 0.5 kGy can reduce microbial counts substantially to improve microbial shelf life without adverse effects on sensory qualities.