Expression of bovine beta-lactoglobulin/human erythropoietin fusion protein in the milk of transgenic mice and rabbits

We have generated several transgenic mouse lines and rabbits expressing efficiently (up to 0.3 mg/ml in mice and up to 0.5 mg/ml in rabbits) human erythropoietin in their milk as bovine beta-lactoglobulin fusion protein. Human erythropoietin cDNA was inserted in frame into exon 5 of the bovine beta-lactoglobulin gene with a linker oligonucleotide encoding the cleavage site for bacterial IgA protease. RNA analysis performed on one lactating transgenic mouse and one transgenic rabbit revealed that the fusion gene was expressed almost exlusively in the mammary gland, although low amounts of transgene-derived RNA were detectable in salivary glands and uterus or in the kidney. The fusion protein was specifically cleaved with IgA protease. The erythropoietin part obtained upon digestion had a lower molecular mass than recombinant erythropoietin produced in Chinese hamster ovary cells. By deglycosylation analysis it was shown that the difference in size was due to a different type of glycosylation. Biological activity of the fusion protein, as determined by growth stimulation of TF-1 erythroleukemia cells, was less than 15% of that of human recombinant erythropoietin. Upon digestion of the fusion protein with IgA protease, biological activity comparable to that of the recombinant erythropoietin was recovered. Transgenic males and virgin females did not show signs of enhanced erythropoiesis, but lactating females expressing the transgene displayed transient increases in their hematocrit values.     

Expression of matrix metalloproteinases and their inhibitors in aneurysms and normal aorta

BACKGROUND: Abdominal aortic aneurysms (AAAs) are characterized by degradation of collagen and elastin resulting from increases in matrix metalloproteinase (MMP) activity. Previous authors have identified isolated increases in expression of specific MMPs in AAAs, but none have compared relative levels of expression of particular MMPs to one another or to those of their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). This study proposes to quantify relative mRNA levels for interstitial collagenase (MMP-1), 72 kd type IV collagenase (MMP-2), 92 kd type IV collagenase (MMP-9), TIMP-1, and TIMP-2 in normal aorta (NA) and AAA to provide insight as to the relative importance of each in aneurysm formation. METHODS: Competitive polymerase chain reactions (PCRs) with gene-specific external standards and cDNA derived from AAAs (n = 8; mean age, 67.4 years) and NA (n = 5; mean age, 40.6 years) were used to quantify mRNA levels. Results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels, determined by means of competitive PCR, and compared by means of Mann-Whitney statistics. RESULTS: Significant increases in MMP mRNA expression in AAA over NA were observed for MMP-1 (3.64 versus 0.3, p = 0.007), MMP-9 (78.03 versus 3.35, p = 0.003), TIMP-1 (835.32 versus 477.2, p = 0.027), and TIMP-2 (18.09 versus 4.14, p = 0.003). The ratio of MMP to TIMP mRNA levels was higher in AAA than NA (0.135 versus 0.045, p = 0.018). CONCLUSIONS: Increases in expression of MMP-1, MMP-9, and MMP/TIMP ratios may result in increased proteolysis and matrix degradation, which characterize AAAs. MMP-9 appears to be the predominant metalloproteinase expressed in AAA, because its mRNA levels were more than 20 times and 2 times higher than those of MMP-1 and MMP-2, respectively. TIMP-1 mRNA levels were in molar excess to those of any of the metalloproteinases studied.     

Solar/UV-induced photocatalytic degradation of three commercial textile dyes.

The photocatalytic degradation of three commercial textile dyes with different structure has been investigated using TiO(2) (Degussa P25) photocatalyst in aqueous solution under solar irradiation. Experiments were conducted to optimise various parameters viz. amount of catalyst, concentration of dye, pH and solar light intensity. Degradation of all the dyes were examined by using chemical oxygen demand (COD) method. The degradation efficiency of the three dyes is as follows: Reactive Yellow 17(RY17) > Reactive Red 2(RR2) > Reactive Blue 4 (RB4), respectively. The experimental results indicate that TiO(2) (Degussa P25) is the best catalyst in comparison with other commercial photocatalysts such as, TiO(2) (Merck), ZnO, ZrO(2), WO(3) and CdS. Though the UV irradiation can efficiently degrade the dyes, naturally abundant solar irradiation is also very effective in the mineralisation of dyes. The comparison between thin-film coating and aqueous slurry method reveals that slurry method is more efficient than coating but the problems of leaching and the requirement of separation can be avoided by using coating technique. These observations indicate that all the three dyes could be degraded completely at different time intervals. Hence, it may be a viable technique for the safe disposal of textile wastewater into the water streams.     

Methods to compare dissolution profiles and a rationale for wide dissolution specifications for metoprolol tartrate tablets

The objectives of this work were to apply several profile comparison approaches to dissolution data of four different but bioequivalent metoprolol tartrate tablet formulations to (1) identify the advantages and disadvantages of each approach, (2) quantify the metric for comparing dissolution profiles of each method, (3) determine metric limits that are consistent with the observed bioequivalence, and (4) rationalize the observed metric limits with respect to the role of dissolution in overall metoprolol absorption. Dissolution was performed by the USP monograph method on four formulations of metoprolol tartrate tablets (Lopressor plus fast, medium, and slow dissolving test formulations). Three general approaches to compare dissolution profiles were examined; they were ANOVA-based, model-independent, and model-dependent approaches. It is concluded that model-independent approaches and several model-dependent approaches yielded numerical results that can serve as objective and quantitative metrics for comparing entire dissolution profiles of the four metoprolol tartrate formulations. However, these methods presented complications. Some metrics were dependent on the length of the dissolution profile and the sampling scheme. Results from the pairwise procedures also depended on the pairing assignment of individual profiles. In spite of complications, these methods suggested wide dissolution specification limits. Wide dissolution specifications were rationalized through an analysis of in vitro-in vivo relationships, which indicated metoprolol dissolution from these formulations was not the rate-limiting step; hence, a range of dissolution profiles can be expected to yield equivalent plasma profiles.     

Oxygenation in the rabbit myocardium: assessment with susceptibility-dependent MR imaging

PURPOSE: To determine the feasibility of using hemoglobin (Hb) desaturation as an indicator of myocardial oxygenation. MATERIALS AND METHODS: High-resolution gradient-echo nuclear magnetic resonance (MR) images of isolated, blood-perfused rabbit hearts were obtained at various blood oxygenation levels. The hearts were perfused at 37 degrees C with a Langendorff apparatus modified for nuclear MR imaging. The perfusate contained bovine red blood cells in a cardioplegic solution that eliminated motion artifacts and minimized arteriovenous oxygenation differences. Hb saturation was varied (7%-100%) randomly. Perfusion pressure was continuously monitored, and blood samples were obtained. RESULTS: There was a substantial correlation between image signal intensity in the myocardium and Hb saturation in the blood, believed to be due to susceptibility effects of the paramagnetic species deoxyhemoglobin. CONCLUSION: Direct and noninvasive determination of regional Hb saturation with susceptibility-dependent MR imaging may provide information regarding regional myocardial O2 content.     

A functional model of the electrical potential of skin

A model of the skin electrical potential has been developed. It contains electrical and functional parameters of the sweating and their dependence on efferent innervation. The analytical expression of the skin electrical potential which explains basic experimental results has been constructed.     

A KBES for integrated engineering

The expert process of engineering involves application of knowledge fundamental to the engineering process at one end, and of compiled knowledge in the form of heuristics gained through many years of experience at the other. Developments in Artificial Intelligence have made the process of computer aided engineering richer by enabling the simulation of this human process of engineering, which until now has been difficult to capture. Knowledge Based Expert Systems (KBES) in engineering developed so far have addressed only narrow domains of the entire process of engineering. This paper discusses a KBES shell (IES) that integrates various AI technologies, which makes it possible to implement new problem-solving strategies required for efficient handling of the entire engineering process. Issues in the integrated process of engineering are discussed first, in order to evolve a specification for the shell. The blackboard architecture is shown to be the ideal backbone for such a shell. The details of inference techniques, the knowledge based backtracking mechanism and the DBMS used in the IES are presented. The application of the IES is demonstrated using the domain of integrated engineering of steel industrial structures as an example. This application encompasses the engineering activities of conceptual design, detailed design, documentation and construction planning.     

Necrotizing fasciitis of the face

Necrotizing fasciitis (NF) is a rapidly progressive soft tissue infection with high morbidity and mortality rates. Its occurrence in the head and neck region is uncommon, the majority of reported cases being limited to involvement of the neck, usually from infections of dental or pharyngeal origin. Involvement of the face from NF is rare; only 35 such cases have been reported in the literature since 1960. It is not only associated with a high mortality but can also result in severe disfigurement of the face, posing challenging reconstructive problems. Successful management of facial NF requires early diagnosis, prompt institution of broad spectrum antibiotics, aggressive surgical debridement to control the infection, and reconstruction of the resultant soft tissue defects. This report describes four additional cases of facial necrotizing fasciitis. One of the four patients died as a result of sepsis and multi-organ system failure. Two of the three surviving patients had significant facial disfigurement. A comprehensive review of the facial NF cases reported in the literature is also provided. Based on our experience with facial NF and the results of all previous case reports, the clinical manifestations, pathogenesis, and management of this disease are discussed.     

Human gamma-interferon expression in the mammary gland of transgenic mice

Transgenic mice carrying a hybrid gene consisting of ovine beta-lactoglobulin gene sequences and human gamma-interferon (hIFN-g) cDNA were produced. hIFN-g expression in the mammary gland of two lactating transgenic founder females was found. The concentration of active hIFN-g in the milk was estimated as being ca. 1800 IU/ml. The hIFN-g ability to express in the mammary gland was found in the progeny of transgenic founder male.