Removal of dihydroxy-terminated components from monomethoxy-terminated poly(ethylene glycol)

A preparative method to remove dihydroxy-terminated components in a sample of presumably monomethoxy, monohydroxy-terminated poly(ethylene glycol) (PEG) is presented. Purification of the monomethoxy-terminated component allows one to prepare a diblock copolymer of PEG and poly(lactic acid) (PLLA) free of a PLLA-PEG-PLLA triblock copolymer in various biomedical applications of the copolymer. Efficiency of the purification is compared for high osmotic pressure chromatography (HOPC) and preparative size exclusion chromatography (SEC). In HOPC, various types of porous silica particles, surfaces, solvents, polymer concentrations have been screened for the optimal performance. It was found that HOPC is more efficient than SEC, especially HOPC of 30-40 wt% solutions in water by a column packed with acid-washed controlled pore glass is optimal in producing high-purity fractions.     

Educational benefits of diversity in medical school: a survey of students.

PURPOSE: Many U.S. medical schools have abandoned affirmative action, limiting the recruitment and reducing the admission of underrepresented minority (URM) students even though research supports the premise that the public benefits from an increase in URM physicians and that URM physicians are likely to serve minority, poor, and Medicaid populations. Faculty and students commonly assume they benefit from peer cultural exchange, and the published evidence for the past two decades supports this notion. This research examined the students' perceptions of the educational merits of a diverse student body by surveying medical students at two schools. METHOD: In 2000, medical students from all four years at Harvard Medical School and the University of California, San Francisco, School of Medicine were enrolled in a telephone survey about the relevance of racial diversity (among students) in their medical education. Students responded to the interviewer's questions on a five-point Likert-type scale. RESULTS: Of the 55% of students who could be located, 97% responded to the survey. Students reported having little intercultural contact during their formative years but significantly more interactions during higher education years, especially in medical school. Students reported contacts with diverse peers greatly enhanced their educational experience. They strongly supported strengthening or maintaining current affirmative action policies in admissions. The responses and demography of the Harvard and UCSF students did not differ significantly, nor did they differ for majority students and URM students-all groups overwhelmingly thought that racial and ethnic diversity among their peers enhanced their education. CONCLUSIONS: Diversity in the student body enhanced the educational experiences of students in two U.S. medical schools.     

Mechanism of adenosine 3′,5′-monophosphate (cAMP)-induced increase in Ca2+ uptake by the sarcoplasmic reticulum in functionally skinned myocardial fibers

The increased rate of Ca²⁺ uptake and ATPase activity in isolated cardiac sarcoplasmic reticulum (SR) by adenosine 3,5-monophosphate (cAMP) has been shown to be activated by a cAMP-dependent protein kinase (cAMP kinase). Functionally skinned myocardial fiber preparations were used to study the mechanisms of cAMP action on the SR at the same time that tension was monitored. cAMP effects were studied on Ca²⁺-activated tension of the contractile proteins, and on Ca²⁺ uptake and release from the SR using caffeine-induced tension transients. Neither cyclic AMP (0.1–5 M) nor the catalytic subunit of cAMP kinase (0.1–1 M) (PK-C) significantly changed either the maximal or the submaximal Ca²⁺-activated tension. The areas of the tension transients were unchanged when cAMP was present in the releasing solution (release phase), and were significantly increased up to a mean of about 80% when cAMP or PK-C was present in the Ca²⁺ loading solutions (uptake phase). The increased tension transient was blocked by the heat-stable inhibitor of cAMP kinase. We conclude that cAMP-induced increases in Ca²⁺ uptake by the SR could play an important role in the positive inotropic effect. cAMP kinase could thus play a crucial role in the regulation of myocardial contractility.     

AMPK as a metabolic switch in rat muscle,liver and adipose tissue after exercise

An increasing body of evidence has revealed that activation of adenosine monophosphate (AMP)‐activated protein kinase (AMPK)‐activated protein kinase increases fatty acid oxidation by lowering the concentration of malonyl coenzyme A (CoA), an inhibitor of carnitine palmitoyl transferase 1. Studies carried out primarily in skeletal muscle suggest that AMPK modulates the concentration of malonyl CoA by concurrently phosphorylating and inhibiting acetyl CoA carboxylase (ACC), the rate limiting enzyme in malonyl CoA synthesis, and phosphorylating and activating malonyl CoA decarboxylase (MCD), an enzyme involved in its degradation. We have recently observed that AMPK and MCD activities are increased and ACC activity diminished in skeletal muscle, liver and, surprisingly, in adipose tissue 30 min following exercise (treadmill run) in normal rats. In liver and adipose tissue these changes were associated with a decrease in the activity of glycerol‐3‐phosphate acyltransferase (GPAT), which catalyses the first committed reaction in glycerolipid synthesis and, which like ACC, is phosphorylated and inhibited by AMPK. Similar changes in ACC, MCD and GPAT were observed following the administration of 5‐aminoimidazole 4‐carboxamide‐riboside (AICAR), further indicating that the exercise‐induced alterations in these enzymes were AMPK‐mediated. Conclusions: (1) AMPK plays a major role in regulating lipid metabolism in multiple tissues following exercise. (2) The net effect of its activation is to increase fatty acid oxidation and diminish glycerolipid synthesis. (3) The relevance of these findings to the regulation of muscle glycogen repletion in the post‐exercise state and to the demonstrated ability of AMPK activation to decrease adiposity and increase insulin sensitivity in rodents remains to be determined.     

Characterization of monoclonal antibody CIBCNSH3 generated to the human EGF receptor

Monoclonal antibody CIBCNSH3 of IgG1 isotype has been generated against human epidermal growth factor receptor (EGFR) using MDA MB 468 breast carcinoma cell line as immunogen. Earlier studies have revealed that this MAb blocked growth factor-receptor interaction and thus inhibited cell proliferation and tumor growth. In the present paper, this MAb has been extensively characterized to evaluate its application in the study of human cancers. The results were compared with those obtained using a control MAb ICR 62 specific to EGFR. Competitive assay showed that this MAb bound to an epitope in the extracellular domain of the EGFR to which MAb ICR 62 also bound. This MAb immunoprecipitated the 170 kD glycoprotein. The specificity was further confirmed by the formation of a single discrete band in western blot analysis. By flow cytometric analysis this monoclonal antibody revealed high binding affinity with MDA MB 468 cells. By immunocytochemical assay, out of 35 breast tumors studied, 40% were found to exhibit strong cell membrane staining and in the case of 25 oral cancers studied, 56% were strong positive. High expression of EGFR was observed in MDA MB 468 cells and HN 5 cells. These studies clearly indicate that MAb CIBCNSH3 might prove useful to identify tumors with high level of expression of EGFR associated with poor prognosis.     

Amplification of fluorescent in situ hybridisation signals in formalin fixed paraffin wax embedded sections of colon tumour using biotinylated tyramide.

Fluorescent in situ hybridisation (FISH) is a powerful tool for the evaluation of chromosomal alterations in formalin fixed paraffin wax embedded sections of colorectal cancer. However, initial experiments using a two-step detection system for digoxigenin labelled chromosome specific centromeric probes resulted in a complete lack of hybridisation signal from a number of colorectal tumour sections. This was due to high levels of background autofluorescence observed in this tissue, which masked any relatively weak hybridisations present. To overcome this problem, a biotinylated tyramide mediated amplification system was incorporated into the FISH detection protocol. This involves the use of horseradish peroxidase to activate the biotinylated tyramide, resulting in the deposition of a large number of biotin molecules at the site of bound peroxidase, which corresponds directly to the location of hybridised probe. Final detection was by means of a streptavidin-FITC conjugate. Using this technique, a panel of 11 colorectal tumour samples studied to date have shown strong, specific hybridisation signals to the nucleus of tumour cells. Amplification of FISH signals by biotinylated tyramide has the potential to improve weak hybridisation signals in cells from numerous sources, using a variety of probe types, including single copy gene probes as well as centromere specific probes.     

Monocyte/macrophage activation by normal bacteria and bacterial products: implications for altered epithelial function in Crohn's disease

Intestinal immune cells are less reactive than those in the peripheral blood; however, such cells from patients with Crohn's disease may be more responsive to bacterial products. Our study examined if nonpathogenic bacteria or lipopolysaccharide (LPS), can affect epithelial function in the presence of monocytes/macrophages. Lamina propria mononuclear cells (LPMCs) and peripheral blood monocytes (PBMs) were obtained from patients with Crohn's disease and control patients. Filter-grown T84 epithelial monolayers were co-cultured with nonactivated or LPS-activated LPMCs or PBMs for 48 hours. Epithelial secretory [baseline short-circuit current (Isc) and DeltaIsc to forskolin] and barrier (transepithelial electrical resistance) parameters were measured in Ussing chambers. LPS-activated PBMs from both controls and patients with Crohn's disease significantly increased Isc ( approximately 300%) and reduced transepithelial electrical resistance ( approximately 40%). Epithelial function was not altered after co-culture with control LPMCs +/- LPS. However, LPMCs from patients with Crohn's disease spontaneously secreted tumor necrosis factor-alpha, and induced epithelial changes similar to those produced by LPS-activated PBMs. Co-culture with control Escherichia coli and PBMs induced comparable changes in epithelial physiology, which were abrogated by anti-tumor necrosis factor-alpha antibody. We conclude that LPMCs of patients with Crohn's disease are spontaneously activated, possibly by gram-negative luminal bacteria, and can directly cause significant alterations in epithelial ion transport and barrier functions.     

A comparison of fluorescein isothiocyanate and lissamine rhodamine (RB 200) as labels for antibody in the fluorescent antibody technique

The relative merits of fluorescein isothiocyanate (FITC) and lissamine rhodamine (RB 200) as labels for antibody in fluorescence microscopy were studied and compared by microphotometry, testing each fluorochrome under its own optimal conditions as far as possible, and at a similar range of dye:protein ratios. The antibody was sheep anti-human globulin, and the tissues stained with it were rat liver sections bearing human anti-nuclear factor on the nuclei. The findings were as follows:

(i) the amount of RB 200 conjugating with protein was strictly proportional to the amount of the sulphonyl chloride derivative added to the reaction mixture; with increasing amounts of FITC in the reaction mixture, however, there was a less than proportional increase in the degree of conjugation.

(ii) Diethylaminoethyl (DEAE)-cellulose chromatography decreased the dye:protein ratio of the conjugates by 40% uniformly for both RB 200 and FITC, regardless of the initial dye:protein ratio.

(iii) When corrections were made for spectral responses of photo-detectors, effects of optimizing the mountants, and benefits to rhodamine of changing from a Xenon to a mercury lamp, it was concluded that RB 200 conjugates could give brighter staining than FITC conjugates at similar dye:protein ratios.

(iv) DEAE-cellulose chromatography greatly improved the contrast of the staining, especially with RB 200 conjugates.

(v) After chromatography, RB 200 consistently gave better contrast than FITC.

(vi) The fluorescence of rhodamine-stained sections did not fade demonstrably when irradiated for several minutes with green light.

(vii) The fluorescence of FITC-stained sections faded rapidly when irradiated with ultra-violet (u.v.)+blue light. The fluorescence appeared to contain two components, one fading with first-order kinetics with a half-life of about a minute under the experimental conditions used and the other not fading at all.

(viii) Raising the pH improved the fluorescence of FITC-stained sections but did not affect rates of fading.

(ix) Narrow-band excitation of FITC-stained sections with blue light instead of u.v.+blue reduced the rate of fading and the fluorescence intensity by equal amounts, an effect presumably due merely to loss of excitation intensity.