Correction of 13C mass isotopomer distributions for natural stable isotope abundance

Metabolism of singly or multiply 13C-labeled substrates leads to the production of molecules that contain 13C atoms at various positions. Molecules differing only in the number of isotopic atoms incorporated are referred to as mass isotopomers. The distribution of mass isotopomers of many molecules can be measured by gas chromatography/ mass spectrometry after chemical derivatization. Quantification of metabolite mass isotopomer abundance resulting from biological processes necessitates correction of the measured mass isotopomer distribution of the derivatized metabolite for contributions due to naturally occurring isotopes of its elements. This correction must take into account differences in the relative natural abundance distribution of each mass isotopomer (skewing). An IBM-compatible computer program was developed which (i) calculates the natural abundance mass isotopomer distribution of unlabeled and labeled standards given the molecular formula of the derivatized molecule or fragment ion, and (ii) calculates the natural abundance mass isotopomer distribution of the singly and multiply labeled molecule or fragment via non-linear fitting to the measured mass isotopomer distribution of the unlabeled molecule or fragment. The output of this program is used to correct measured mass isotopomer distributions for contributions from natural isotope abundances and to verify measured values for theoretical consistency. Differences between predicted and measured unlabeled and 13C-labeled isotopomer distributions for hydroxamate di-t-butyl-dimethylsilyl (di-TBDMS) derivatized pyruvate were measured. The program was applied to the mass isotopomer distribution of glucose labeled from [U-13C3]glycerol and of fatty acids labeled from [U-13C6]glucose and either [2-13C2] acetate or [U-13C2]acetate. In some of these cases, the measured mass isotopomer distributions corrected by the program were different from those corrected by the classical technique. Implications of these differences including those on the calculation of glucose production due to gluconeogenesis in isolated perfused rat liver are discussed.     

Alteration of the pharmacokinetics of small proteins by iodination

The pharmacokinetics of several proteins were investigated using two different assays. A 23 kDa recombinant protein fragment of bactericidal/permeability-increasing protein (rBPI23) was radiolabeled with 125I using Iodo-beads and administered rats. Plasma samples were collected and assayed for 125I-rBPI23 by radioactivity. In a separate experiment, rBPI23 was administered to rats and plasma samples were assayed for rBPI23 by ELISA. The clearance determined from plasma concentrations of 125I-rBPI23 measured by radioactivity was about 2.5-fold lower than that of rBPI23 determined by ELISA. In addition, the steady state volumes of distribution and mean residence times of 125I-rBPI23 measured by radioactivity were four-fold and 10-fold greater, respectively, compared to those measured by the ELISA method. By studying several proteins with a range of molecular weights, we found that the pharmacokinetics of proteins below about 60 kDa were different when assayed by radioactivity or ELISA, but those of proteins with molecular weights of at least 80 kDA revealed only minor differences. To determine which assay method yielded the correct plasma pharmacokinetic profile, rBPI23 was metabolically labeled with 35S-methionine and administered to rats, and plasma samples were assayed by radioactivity. The concentration-time profile assessed by this method was very close to that determined by ELISA. Exposing rBPI23 to chloramine-T (the oxidant used in the iodination process) and measuring its plasma concentration by ELISA revealed pharmacokinetics similar to those of the iodinated protein measured by radioactivity. In contrast, radiolabeling rBPI23 using iodinated Bolton-Hunter reagent (which avoids exposing the protein to oxidant), and measuring 125I-rBPI23 by radioactivity, yielded pharmacokinetics that were similar, although not identical, to the pharmacokinetics of rBPI23 measured by ELISA. Thus, our data suggest that directly iodinating low-molecular-weight proteins by oxidation procedures alters their clearance from the blood, preventing reliable determination of pharmacokinetic parameters.     

Ceramide induces hepatocyte cell death through disruption of mitochondrial function in the rat

Although ceramide signaling pathways have been implicated in cell death, neither their role in hepatocellular death nor the cellular mechanisms mediating ceramide-induced cell death are known. The mitochondrial membrane permeability transition (MMPT) has been proposed as a common final pathway in cell death. Thus the aims of our study were to determine if ceramides cause hepatocellular death by necrosis and not apoptosis as confirmed by morphology and the absence of internucleosomal DNA cleavage. Ceramide-mediated hepatocyte necrosis was acyl chain-length, concentration, and time-dependent. Ceramides induced cell necrosis was associated with adenosine triphosphate (ATP) depletion and mitochondrial depolarization suggesting that ceramides caused mitochondrial dysfunction. In isolated mitochondria, ceramides induced the cyclosporine A-sensitive MMPT in an acyl chain-length and concentration dependent manner. Ceramide toxicity was specific as the less potent dihydro form did not induce cell necrosis, significant ATP depletion, mitochondrial depolarization nor the MMPT. In conclusion, ceramide induced cell death is acyl-chain length dependent and mediated by the MMPT. These data show for the first time that ceramide acts as a mediator of hepatocyte necrosis by causing mitochondrial failure.     

Conscious neurosensory mapping of the internal structures of the human knee without intraarticular anesthesia

The conscious neurosensory characteristics of the internal components of the human knee were documented by instrumented arthroscopic palpation without intraarticular anesthesia. With only local anesthesia injected at the portal sites, the first author (SFD) had both knees inspected arthroscopically. Subjectively, he graded the sensation from no sensation (0) to severe pain (4), with a modifier of either accurate spatial localization (A) or poor spatial localization (B). The nature of the intraarticular sensation was variable, ranging from 0 on the patellar articular cartilage to 4A on the anterior synovium, fat pad, and joint capsule. The sensation arising from the cruciate ligaments ranged from 1 to 2B in the midportion, and from 3 to 4B at the insertion sites. The sensation from the meniscal cartilages ranged from 1B on the inner rim to 3B near the capsular margin. Innervation of most intraarticular components of the knee is probably crucial for tissue homeostasis. Failure of current intraarticular soft tissue reconstructions of the knee may be due, in part, to the lack of neurosensory restoration. Research studies of the knee designed to delineate factors that restore neurosensory characteristics of the musculoskeletal system may lead to techniques that result in true restoration of joint homeostasis and function.     

CCR5 promoter polymorphism and HIV-1 disease progression. Multicenter AIDS Cohort Study (MACS)

BACKGROUND: The rate of progression to AIDS varies among individuals infected with HIV-1. Factors responsible include two inherited human alleles, CCR5 delta32 and CCR2-641, which alter the protein-coding regions for the HIV-1 coreceptors/chemokine receptors CCR5 and CCR2b. We tested the hypothesis that polymorphisms of the CCR5 promoter might affect the rate of progression of HIV-1 infected people to AIDS. METHODS: We used directed heteroduplex analysis to identify polymorphism in the CCR5 promoter. Promoter-variants were compared in vitro with a chloramphenicol acetyltransferase reporter gene, and in vivo by genotyping HIV-1 seroconvertors discordant at polymorphous loci. FINDINGS: An A/G polymorphism was identified at basepair 59029 (Genbank U95626) in the CCR5 promoter. Both promoter alleles were common (43-68% allelic frequency for 59029-A depending on race). When in-vitro promoter activity was measured, 59029-G had 45% lower activity than 59029-A (p=0.05). In a cohort of HIV-1 seroconvertors lacking both CCR5 delta32 and CCR2-641, 59029-G/G individuals progressed to AIDS on average 3.8 years more slowly than 59029-A/A individuals (p=0.004). 59029-G/A discordance did not correlate with discordant rates of infection. INTERPRETATION: Our results are consistent with the hypothesis that CCR5 is important in HIV-1 pathogenesis. CCR5 59029-G/G appears to be protective relative to CCR5 59029-A/A, and about twice as protective relative to CCR5 delta32 or CCR2-641. This effect may be the result of reduced CCR5 mRNA production. These results identify the first site in the CCR5 promoter that may be a useful target for treatment of HIV-1 infection.