Radiotherapy: effects on expanded skin

Although the combination of radiation and tissue expansion has been associated with a significant rate of complications, the specific pathophysiology has yet to be clearly elucidated. The objective of this study was to develop a model to identify and examine specific histologic changes associated with tissue expansion and irradiation. Rectangular 50-cc silicone tissue expanders were placed subcutaneously over the midline dorsum of 18 adult New Zealand white rabbits. Preoperative radiographic dosimetry demonstrated that the radiation portal was away from vital intraabdominal structures. The expanders were inflated with 10 cc of saline every other day for a total of 80 cc. Expanders were left in place for 2 to 3 weeks to allow fibrovascular capsule formation. The rabbits were then divided into three groups (six rabbits per group), each receiving one of three nonfractionated doses of radiation (20, 25, or 35 Gy). Half of the expanded skin was irradiated using a single dose, and the other half served as a nonirradiated control. Capsules and skin were harvested 6 weeks after the delivery of radiation, allowing the beginning of chronic radiation changes to occur. Using hematoxylin and eosin staining, histomorphometric analysis was performed. The data were analyzed using Student's test. Although irradiation did not affect dermal thickness, it did cause a statistically significant increase in epidermal thickness. At 20, 25, and 35 Gy the increase in epidermal thickness was 43, 90, and 130 percent, respectively. Although significant epidermal changes could be identified, capsular and dermal alterations were not evident. Further studies evaluating the long-term effects of alterations in capsular formation caused by radiation may be required.     

Continuing to break the sound barrier: genes in hearing

The past year has seen major advances in our understanding of the genes involved in Usher syndrome, as well as the discovery of a myriad of other genes expressed specifically in hair cells. Mouse models continue to be invaluable in illuminating our knowledge of how mutations in genes lead to deafness. The role of mitochondrial genes in the hearing process has also contributed to elucidating the workings of the auditory system.     

CCR3 antagonists: a potential new therapy for the treatment of asthma. Discovery and structure-activity relationships

CCR3 antagonist leads with IC(50) values in the microM range were converted into low nM binding compounds that displayed in vitro inhibition of human eosinophil chemotaxis induced by human eotaxin. In particular, 4-benzylpiperidin-1-yl-n-propylureas and erythro-3-(4-benzyl-2-(alpha-hydroxyalkyl)piperidin-1-yl)-n-propylureas (obtained via Beak reaction of N-BOC-4-benzylpiperidine) exhibited single digit nanomolar IC(50) values for CCR3.     

Characterization of a novel parainfluenza virus,caviid parainfluenza virus 3, from laboratory guinea pigs (Cavia porcellus)

A novel Respirovirus was isolated from nasopharyngeal swab specimens from clinically normal laboratory guinea pigs, and was characterized and named caviid parainfluenza virus 3 (CavPIV-3). The CavPIV-3 is enveloped, is 100 to 300 nm in diameter, and has a characteristic 15-nm-diameter chevron-shaped virus ribonucleocapsid protein. Sequence analysis of the fusion glycoprotein of CavPIV-3 revealed it to be 94% identical to human and guinea pig parainfluenza 3 (PIV-3) viruses and 80% identical to bovine PIV-3. To determine whether CavPIV-3 causes clinical disease in laboratory guinea pigs and to compare the serologic response of guinea pigs to CavPIV-3 and to other paramyxoviruses, an infection study was performed, in which groups of guinea pigs were inoculated with CavPIV-3, Sendai virus, simian virus 5 (SV-5), murine pneumonia virus (PVM), or bovine PIV-3 virus. During the course of the study, guinea pigs were maintained in an infectious disease suite, housed in Micro-Isolator cages, and were only manipulated under a laminar flow hood. Clinical signs of disease were not observed in any of the paramyxovirus-inoculated guinea pigs during the eight-week course of the study, and histologic signs of disease were not evident at necropsy eight weeks after inoculation. Guinea pigs inoculated with CavPIV-3, Sendai virus, PVM, and bovine PIV-3 developed robust homologous or heterologous serologic responses. In contrast, guinea pigs inoculated with SV-5 developed modest or equivocal serologic responses, as assessed by use of an enzyme-linked immunosorbent assay. Further, use of the SV-5 enzyme-linked immunosorbent assay resulted in the highest degree of non-specific reactivity among all of the paramyxovirus assays. In summary, CavPIV-3 is a novel guinea pig Respirovirus that subclinically infects laboratory guinea pigs, resulting in a robust serologic response, but no observed clinical or histologic disease. The CavPIV-3 fusion glycoprotein gene sequence is available from GenBank as accession No. AF394241, and the CavPIV-3 virus is available from the American Type Culture Collection as accession No. DR-1547.     

Full subunit coverage liquid chromatography electrospray ionization mass spectrometry (LCMS+) of an oligomeric membrane protein: cytochrome b(6)f complex from spinach and the cyanobacterium Mastigocladus laminosus

Highly active cytochrome b(6)f complexes from spinach and the cyanobacterium Mastigocladus laminosus have been analyzed by liquid chromatography with electrospray ionization mass spectrometry (LCMS+). Both size-exclusion and reverse-phase separations were used to separate protein subunits allowing measurement of their molecular masses to an accuracy exceeding 0.01% (+/-3 Da at 30,000 Da). The products of petA, petB, petC, petD, petG, petL, petM, and petN were detected in complexes from both spinach and M. laminosus, while the spinach complex also contained ferredoxin-NADP(+) oxidoreductase (Zhang, H., Whitelegge, J. P., and Cramer, W. A. (2001) Flavonucleotide:ferredoxin reductase is a subunit of the plant cytochrome b(6)f complex. J. Biol. Chem. 276, 38159-38165). While the measured masses of PetC and PetD (18935.8 and 17311.8 Da, respectively) from spinach are consistent with the published primary structure, the measured masses of cytochrome f (31934.7 Da, PetA) and cytochrome b (24886.9 Da, PetB) modestly deviate from values calculated based upon genomic sequence and known post-translational modifications. The low molecular weight protein subunits have been sequenced using tandem mass spectrometry (MSMS) without prior cleavage. Sequences derived from the MSMS spectra of these intact membrane proteins in the range of 3.2-4.2 kDa were compared with translations of genomic DNA sequence where available. Products of the spinach chloroplast genome, PetG, PetL, and PetN, all retained their initiating formylmethionine, while the nuclear encoded PetM was cleaved after import from the cytoplasm. While the sequences of PetG and PetN revealed no discrepancy with translations of the spinach chloroplast genome, Phe was detected at position 2 of PetL. The spinach chloroplast genome reports a codon for Ser at position 2 implying the presence of a DNA sequencing error or a previously undiscovered RNA editing event. Clearly, complete annotation of genomic data requires detailed expression measurements of primary structure by mass spectrometry. Full subunit coverage of an oligomeric intrinsic membrane protein complex by LCMS+ presents a new facet to intact mass proteomics.     

Measurement error affects risk estimates for recruitment to the Hudson River stock of striped bass

We examined the consequences of ignoring the distinction between measurement error and natural variability in an assessment of risk to the Hudson River stock of striped bass posed by entrainment at the Bowline Point, Indian Point, and Roseton power plants. Risk was defined as the probability that recruitment of age-1+ striped bass would decline by 80% or more, relative to the equilibrium value, at least once during the time periods examined (1, 5, 10, and 15 years). Measurement error, estimated using two abundance indices from independent beach seine surveys conducted on the Hudson River, accounted for 50% of the variability in one index and 56% of the variability in the other. If a measurement error of 50% was ignored and all of the variability in abundance was attributed to natural causes, the risk that recruitment of age-1+ striped bass would decline by 80% or more after 15 years was 0.308 at the current level of entrainment mortality (11%). However, the risk decreased almost tenfold (0.032) if a measurement error of 50% was considered. The change in risk attributable to decreasing the entrainment mortality rate from 11 to 0% was very small (0.009) and similar in magnitude to the change in risk associated with an action proposed in Amendment #5 to the Interstate Fishery Management Plan for Atlantic striped bass (0.006)--an increase in the instantaneous fishing mortality rate from 0.33 to 0.4. The proposed increase in fishing mortality was not considered an adverse environmental impact, which suggests that potentially costly efforts to reduce entrainment mortality on the Hudson River stock of striped bass are not warranted.     

Structural characterization of monomeric folding intermediates of recombinant human macrophage-colony stimulating factor beta (rhM-CSFbeta) by chemical trapping,chromatographic separation and mass spectrometric peptide mapping

We have developed a strategy for the characterization of protein folding intermediates that combines selective modification of bis-cysteinyl thiol groups with melarsen oxide (MEL), chromatographic separation and mass spectrometric characterization of the resulting protein derivatives. In the unfolding reaction of recombinant human macrophage-colony stimulating-factor beta (rhM-CSFbeta) we observed monomeric M.4MEL and dimeric D.2MEL intermediates. The major locations of the MEL groups in D.2MEL were at C157 and C159. In M.4MEL, MEL groups were predominantly located at C31 and C102. These results indicate the presence of highly structured dimeric and monomeric intermediates. In the completely reduced R.4MEL derivative, MEL groups were distributed such that the smallest ring structures resulted.     

Replication and compartmentalization of HIV-1 in kidney epithelium of patients with HIV-associated nephropathy

HIV-associated nephropathy is a clinicopathologic entity that includes proteinuria, focal segmental glomerulosclerosis often of the collapsing variant, and microcystic tubulointerstitial disease. Increasing evidence supports a role for HIV-1 infection of renal epithelium in the pathogenesis of HIV-associated nephropathy. Using in situ hybridization, we previously demonstrated HIV-1 gag and nef mRNA in renal epithelial cells of patients with HIV-associated nephropathy. Here, to investigate whether renal epithelial cells were productively infected by HIV-1, we examined renal tissue for the presence of HIV-1 DNA and mRNA by in situ hybridization and PCR, and we molecularly characterized the HIV-1 quasispecies in the renal compartment. Infected renal epithelial cells were removed by laser-capture microdissection from biopsies of two patients, DNA was extracted, and HIV-1 V3-loop or gp120-envelope sequences were amplified from individually dissected cells by nested PCR. Phylogenetic analysis of kidney-derived sequences as well as corresponding sequences from peripheral blood mononuclear cells of the same patients revealed evidence of tissue-specific viral evolution. In phylogenetic trees constructed from V3 and gp120 sequences, kidney-derived sequences formed tissue-specific subclusters within the radiation of blood mononuclear cell-derived viral sequences from both patients. These data, along with the detection of HIV-1-specific proviral DNA and mRNA in tubular epithelium cells, argue strongly for localized replication of HIV-1 in the kidney and the existence of a renal viral reservoir.