Protein analysis through Western blot of cells excised individually from human brain and muscle tissue.

Comparing protein levels from single cells in tissue has not been achieved through Western blot. Laser capture microdissection allows for the ability to excise single cells from sectioned tissue and compile an aggregate of cells in lysis buffer. In this study we analyzed proteins from cells excised individually from brain and muscle tissue through Western blot. After we excised individual neurons from the substantia nigra of the brain, the accumulated surface area of the individual cells was 120,000, 24,000, 360,000, 480,000, 600,000 µm2. We used an optimized Western blot protocol to probe for tyrosine hydroxylase in this cell pool. We also took 360,000 µm2 of astrocytes (1700 cells) and analyzed the specificity of the method. In muscle we were able to analyze the proteins of the five complexes of the electron transport chain through Western blot from 200 human cells. With this method, we demonstrate the ability to compare cell-specific protein levels in the brain and muscle and describe for the first time how to visualize proteins through Western blot from cells captured individually.

Reassortment between human A(H3N2) viruses is an important evolutionary mechanism.

Phylogenetic relationships of whole genomes of H3N2 viruses circulating in Germany during a 6-year period from 1998 to 2005 revealed the co-circulation of different lineages of viruses. Multiple reassortment events occurred during this time between viruses belonging to different lineages or different subgroups. Strains isolated during 1998-1999 were characterised by a surprisingly high heterogeneity and multiple reassortment events. Seventy percent of the examined 1998-1999 viruses had completely different genome compositions. To our knowledge, such an exceptional high proportion of different reassortant strains, encompassing all eight genome segments, have not been described before. In contrast, only one reassortant virus was prevalent during 1999-2000 even though two of the three 1998-1999 lineages were co-circulating. Reassortant viruses were isolated also in each of the other seasons. However, the proportion of H3N2 viruses with different genome compositions varied from season to season. Strains with a reassortant NA played an important role and were also detected during 2003-2004 and 2004-2005 accounting for 45% and 70% of the circulating H3N2 viruses, respectively. Moreover, different reassortment events occurring during these seasons included also the PB1, PB2 and NP genes. The results presented here emphasize that genetic reassortment is an important factor in the evolution of H3N2 viruses and highlight the need for a comprehensive analysis of influenza viruses, especially with regard to the annual vaccine composition.

Carvedilol as therapy in pediatric heart failure: an initial multicenter experience.

OBJECTIVE: The objective was to determine the dosing, efficacy, and side effects of the nonselective beta-blocker carvedilol for the management of heart failure in children. STUDY DESIGN: Carvedilol use in addition to standard medical therapy for pediatric heart failure was reviewed at 6 centers. RESULTS: Children with dilated cardiomyopathy (80%) and congenital heart disease (20%), age 3 months to 19 years (n = 46), were treated with carvedilol. The average initial dose was 0.08 mg/kg, uptitrated over a mean of 11.3 weeks to an average maintenance dose of 0.46 mg/kg. After 3 months on carvedilol, there were improvements in modified New York Heart Association class in 67% of patients (P =.0005, chi2 analysis) and improvement in mean shortening fraction from 16.2% to 19.0% (P =.005, paired t test). Side effects, mainly dizziness, hypotension, and headache, occurred in 54% of patients but were well tolerated. Adverse outcomes (death, cardiac transplantation, and ventricular-assist device placement) occurred in 30% of patients. CONCLUSIONS: Carvedilol as an adjunct to standard therapy for pediatric heart failure improves symptoms and left ventricular function. Side effects are common but well tolerated. Further prospective study is required to determine the effect of carvedilol on survival and to clearly define its role in pediatric heart failure therapy.

Primary structure of hepatocyte nuclear factor/forkhead homologue 4 and characterization of gene expression in the developing respiratory and reproductive epithelium.

Members of the winged helix/forkhead family of transcription factors are believed to play a role in cell-specific gene expression. A cDNA encoding a member of this family of proteins, termed hepatocyte nuclear factor/forkhead homologue 4 (HFH-4), has been isolated from rat lung and rat testis cDNA libraries. This cDNA contains an open reading frame of 421 amino acids with a conserved DNA binding domain and several potential transactivating regions. During murine lung development, a single species of HFH-4-specific transcript (2.4 kb long) is first detected precisely at the start of the late pseudoglandular stage (embryonic day 14.5) and, by in situ hybridization, is specifically localized to the proximal pulmonary epithelium. The unique temporal and spatial pattern of HFH-4 gene expression in the developing lung defines this protein as a marker for the initiation of bronchial epithelial cell differentiation and suggests that it may play an important role in cell fate determination during lung development. In addition to expression in the pulmonary epithelium, RNA blot analysis reveals 2.4-kb HFH-4 transcripts in the testis and oviduct. By using mice with genetic defects in spermatogenesis, HFH-4 expression in the testis is found to be associated with the appearance of haploid germ cells and in situ hybridization studies indicate that HFH-4 expression is confined to stages I-VII of spermatogenesis. This pattern of HFH-4 gene expression during the early stages of differentiation of haploid germ cells suggests that HFH-4 may play a role in regulating stage-specific gene expression and cell-fate determination during lung development and in spermatogenesis.

Stability of foscarnet sodium in 0.9% sodium chloride injection.

The stability of foscarnet sodium in 0.9% sodium chloride injection was studied. Foscarnet sodium 24 mg/mL was added to polyvinyl chloride i.v. bags containing 0.9% sodium chloride injection to give a theoretical foscarnet sodium concentration of 12 mg/mL. Ten bags of solution were stored at 25 degrees C in normal laboratory light, 10 bags were stored at 25 degrees C in the dark, and 10 bags were stored at 5 degrees C in the dark. Samples were analyzed for foscarnet concentration by high-performance liquid chromatography on days 0, 3, 8, 10, 15, and 30. The solutions were visually inspected for particulates, and pH was measured. Foscarnet sodium was stable in all the solutions for up to 30 days under all the storage conditions; mean foscarnet concentrations did not decline below 99% of initial values. No particulates appeared, and there were no important changes in pH. Foscarnet sodium 12 mg/mL in 0.9% sodium chloride injection was stable for up to 30 days when stored at 25 degrees C and exposed to light, 25 degrees C and protected from light, or 5 degrees C and protected from light.

Enzymatic sulfation of steroids. XI. The extensive purification and some properties of hepatic sulfotransferase III from female rats.

Our earlier studies showed that livers from female rats contained three glucocorticoid sulfating enzymes we named sulfotransferases I, II, and III, (STI, STII, and STIII, respectively). In this report STIII from female Charles River CD rats was purified 1010- to 1300-fold compared with liver homogenates. The most highly purified STIII fraction electrophoresed as a single protein band. The molecular weight of STIII was 68 300 +/- 4900. Its pH optimum for cortisol sulfation was pH 6.0 +/- 0.1. However, it was routinely assayed at pH 6.8 for reasons enumerated in the text. Cortisol sulfation by STIII proceeded by either an ordered sequential mechanism or by an Iso Theorell-Chance mechanism at pH 6.8. The Km's for cortisol and the reaction coenzyme, 3'-phosphoadenosine-5'-phosphosulfate were 6.48 +/- 0.78 and 6.78 +/- 1.26 microM, respectively. Comparison of the ability of the enzyme to sulfate 40 microM cortisol, estradiol-17 beta, testosterone, deoxycorticosterone, and dehydroepiandrosterone, showed that the glucocorticoid was sulfated preferentially. Interestingly, its cortisol sulfotransferase activity was inhibited by a number of steroids. p-Hydroxymercuribenzoate inhibition studies indicated the presence of essential sulfhydryl groups in STIII. The enzyme was activated by divalent Ba, Ca, Co, Cr, Mg, Mn, and Ni salts. It was inactivated by Zn2+ and Cd2+ salts. The text compares STIII from female rats with other steroid sulfotransferases including the major glucocorticoid sulfotransferase from male rats.

Relationship between radiation response and the deoxyribonucleic acid replication cycle in bacteria: dependence on the excision-repair system.

Prestarvation of Escherichia coli for required amino acids results in a marked enhancement in both ultraviolet light (UV) or X-ray resistance for selective strains. Preventing protein synthesis by starvation for required amino acids results in completion of the cycle of chromosomal replication then underway. We have investigated the relationship between starvation-induced resistance enhancement (SIRE) and the excision-repair (Hcr) system in several E. coli strains including E. coli B/r hcr(+) and its isogenic mutant E. coli B/r hcr(-). The following observations were made. (i) The Hcr system is the major component of SIRE in UV-irradiated strain B/r. By using the Hcr(+) strain, SIRE increases the 10% survival dose from approximately 400 ergs to approximately 1,200 ergs/mm(2). With the Hcr cells, the increase is from approximately 45 ergs to 60 ergs/mm(2). (ii) Although prestarvation leads to a moderate enhancement of resistance to X irradiation, this effect is not dependent on the Hcr system. (iii) The double mutant, E. coli B(s-1) (hcr(-)exr(-)) is completely unable to express SIRE whether studied with UV or X irradiation. It is concluded that the Hcr system is the major system responsible for SIRE in UV-treated cells, whereas Exr (resistance to X rays) may be involved to a minor extent. The Exr character appears to be required for SIRE expression in X-ray exposed cells.

The dampening of DNA replication cycles after x-irradiation or ultraviolet irradiation.

Escherichia coli strains 15T(-) (555-7) and B/r were grown in the presence of thymine-(14)C to label all DNA. The ability of these parental DNA's to undergo cycles of replication subsequent to cellular irradiation with either X-ray or ultraviolet light (UV) was followed with density labels. Exposed cells were shifted into the density medium at times which were approximately multiples of normal rounds of DNA replication. A portion of the parental DNA, replicated semiconservatively once during an initial cycle following UV or X-irradiation in E. coli, failed to replicate again within the time studied. The time course of semiconservative parental DNA replication is altered.