大鼠中枢和外周神经损伤后胞体分布区神经组织的红外光谱分析

目的：观察外周和中枢神经损伤后胞体分布区神经组织总蛋白和核酸的变化规律。方法：应用傅里叶红外光谱(FTIR)检测方法，分析大鼠脊髓和坐骨神经损伤后相应的神经元胞体分布区神经组织中归属为核酸和蛋白质的谱带吸收强度的变化。结果：伤后早期，外周和中枢神经损伤后其相应胞体部位神经组织RNA，DNA和蛋白质的含量均增加；1周后，外周和中枢神经元胞体分布区RNA，DNA和蛋白质含量均变化规律不同：外周神经元胞体RNA和DNA的含量仍较高，而中枢神经伤侧和对照侧胞体RNA和DNA的含量接近，中枢和外周神经元胞体蛋白含量变化相反。结论：外周和中枢神经损伤后再生过程中其胞体反应性不同，这一差异可能与外周和中枢神经损伤后再生差异有关。     

Identification of N2 as a metabolite of acetylhydrazine in the rat

 In the pathogenesis of isoniazid-induced hepatic injury, cytochrome P450-dependent metabolic activation of the metabolite, acetylhydrazine (AcHz), is the crucial step. Exhalation of [¹⁴C]-carbon dioxide has previously been used to quantify indirectly this pathway. In contrast, according to the current concept of AcHz bioactivation, molecular nitrogen is produced directly, but has not yet been identified. Here, we measured [¹⁵N]-nitrogen and ¹⁴CO₂ exhalation, after the administration of [¹⁵N₂]-[¹⁴C]-AcHz, in rats. Laser magnetic resonance (LMR) spectroscopy, a new sensitive and specific technique for the measurement of ¹⁵N and ¹⁴N in gas samples, was used. To demonstrate the involvement of cytochrome P450, rats were treated with phenobarbital (PB) or PB + cobalt(II) chloride (CoCl₂) (n=3 in each group). Time-dependent ¹⁵N₂ exhalation differed significantly between treatment groups (p<0.001). At 240 min, cumulative exhalation of ¹⁵N was 1.92±0.43% (mean±SE) of the dose in the control group, 2.53±0.23% in the PB group, and 1.00±0.15% in the PB+CoCl₂ group (p<0.05 compared to controls, p<0.01 compared to PB). Cumulative exhalation of ¹⁴CO₂ in 24 h ranged from 15.1 to 21.9%, with no significant difference between treatment groups. In conclusion, N₂ is a metabolite of AcHz. N₂ formation reflects the cytochrome P450-mediated activation of AcHz and can be used as an index of this pathway. Generally, LMR spectroscopy is valuable for monitoring any N₂-liberating process in vivo. Received: 14 March 1995/Accepted: 15 August 1995     

Synergistic renal protection by combining alkaline-diuresis with lipid peroxidation inhibitors in rhabdomyolysis: possible interaction between oxidant and non-oxidant mechanisms

BACKGROUND AND PURPOSE.: Heme-proteins, besides causing renal tubular obstruction, maycontribute to rhabdomyolysis-induced renal injury through aheme-iron-mediated lipid peroxidation process. In the presentstudy, we compared the combined therapy of a lipid peroxidationinhibitor, 21-aminosteroid (21-AS) and fluid-alkaline-mannitol(FAM) diuresis with either of them alone to determine the efficacyof the combination therapy and to delineate the roles of lipidperoxidation and cast formation. METHODS AND RESULTS.: Employing Raman spectroscopy, we confirmed in vitro the abilityof 21-AS to inhibit iron-induced fatty acid peroxidation. 21-ASwas then administered to rats developing renal failure fromglycerol-induced rhabdomyolysis. Although 21-AS inhibited rhabdomyolysis-inducedplasma and renal lipid peroxidation, renal protection was incomplete.Administration of FAM to inhibit cast formation afforded a betterrenal protection. However, when these therapies were combinedto inhibit both lipid peroxidation and cast formation, therewas a synergistic renal functional protection. This was accompaniedby a maximum inhibition of renal and plasma lipid peroxidation,as well as, renal tubular necrosis and cast formation. Comparedto combination therapy, FAM therapy alone, despite identicalvolume, was accompanied by a higher tubular necrosis and castformation. CONCLUSIONS.: That combining a lipid peroxidation inhibitor with fluid-alkalinediuresis in rhabdomyolysis further lowers renal lipid peroxidation,tubular necrosis and cast formation and synergistically limitsrenal dysfunction (i) supports a role for lipid peroxidationin the pathophysiology of rhabdomyolysis ARF, (ii) underscoresthe role of intratubular heme retention, a cause for tubularobstruction as well a source for prodigious amount of iron,likely involved in the lipid peroxidation, and (iii) raisesthe possibility of interactions between non-oxidant and oxidantmechanisms.     

The role of Na+/K+ ATPase activity during low flow ischemia in preventing myocardial injury: A 31P, 23Na and 87Rb NMR spectroscopic study

An increase in intracellular Na⁺ during ischaemia has been associated with myocardial injury. In this study, we determined whether inhibition of Na⁺/K⁺ ATPase activity contributes to this increase and whether Na⁺/K⁺ ATPase activity can be maintained by provision of glucose to perfused rat hearts during low flow, 0.5 ml/min, ischemia. We used ³¹P NMR spectroscopy to determine changes in myocardial energetics and intracellular and extracellular volumes. ²³Na NMR spectroscopy, with DyTTHA^3- present as a shift reagent, was used to measure changes in intracellular Na⁺ and ⁸⁷Rb NMR spectroscopy was used to estimate Na⁺/K⁺ ATPase activity from Rb⁺ influx rates, Rb⁺ being an NMR-sensitive congener of K⁺. In hearts provided with 11 mM glucose throughout ischemia, glycolysis continued and ATP was twofold higher than in hearts without glucose. In the glucose-hearts, Rb⁺ influx rate was threefold higher, intracellular Na⁺ was fivefold lower at the end of ischemia and functional recovery during reperfusion was twofold higher. We propose that continuation of glycolysis throughout low flow ischemia allowed maintenance of sufficient Na⁺/K⁺ ATPase activity to prevent the increase in intracellular Na⁺ that would otherwise have led to myocardial injury.     

The distribution of lead in human hemopoietic tissue and spongy bone after lead poisoning and Ca-EDTA chelation therapy

Two iliac crest needle biopsies were taken from a 43-year-old lead-poisoned woman during and after completion of a Ca-EDTA treatment. By atomic absorption spectroscopy the first and second biopsy were found to contain 56, respectively 41.6 g lead/g wet tissue. In both biopsies 36% of the lead was extractable in 0.1 N HCl. Electron microbeam X-ray analysis proved to have too low sensitivity for quantitation of the lead in these biopsies. Laser microbeam mass analysis (LAMMA), performed only on the second biopsy, revealed a high and fairly constant residual lead concentration in all bone marrow cell nuclei (approximately 55 g/g) and a low lead concentration in the cytoplasm of the same cells (4–12 (g/g). The extracellular bone matrix lead was greatly concentrated in the superficial 3–6 m osteoid zone of the bony trabeculae and totally absent from deeper parts of the mineralized matrix. The LAMMA results are in good agreement with those of subcellular fractionation experiments and atomic absorption spectroscopy, provided that the relative volume fraction of nucleus and cytoplasm is accounted for. The high residual osteoid lead after completed chelation therapy indicates that lead has a stronger affinity for the organic than the mineral components of bone matrix.     

Identification of GRASP-1 as a novel 97 kDa autoantigen localized to endosomes

We have identified an autoantigen that is recognized by antibodies from an 18-year-old female with a history of recurrent infections who later in her clinical course developed Raynaud's phenomenon and telangiectasias. By indirect immunofluorescence (IIF), the index serum produced a unique cytoplasmic discrete speckled (CDS) staining pattern that partially colocalized with early endosome antigen 1 (EEA1) but not Golgi complex or other cytoplasmic organelles in HEp-2 cells. When HEp-2 cells were treated with 0.1 N HCl, the cytoplasmic speckled staining of the index serum was markedly decreased, suggesting that the reactive antigen was soluble. Western blot analysis showed a reactive approximately 97 kDa protein in a saline soluble protein preparation from HeLa cells. Mass spectrometric analysis of the excised 97 kDa band that was immunoprecipitated from HeLa cell extracts identified GRASP-1 as a possible target. The index serum and anti-GRASP-1 antibodies colocalized to structures in the cytoplasm of HEp-2 cells. Synthetic peptides representing the full-length GRASP-1 protein were used to identify reactive epitopes. Like many other cytoplasmic autoantigens, GRASP-1 has numerous coiled-coil domains throughout the protein with the exception of short segments at the amino and carboxyl terminus.     

Specifically associated PCR products probed by coincident detection of two-color cross-correlated fluorescence intensities in human gene polymorphisms of methylene tetrahydrofolate reductase at site C677T: a novel measurement approach without follow-up mathematical analysis

Whole blood samples of known methylene tetrahydrofolate reductase (MTHFR) genotypes from 24 individuals were examined at site C677T. Their amplified DNA products were assessed by two-color fluorescence cross-correlation measurements and agarose gel electrophoresis/capillary gel electrophoresis. DNA subpopulations were identified which were not associated with the proper genotype by primer combinations and cycling conditions called multiplexes. We confirmed that DNA analysis by two-color fluorescence cross-correlation measurements allowed the detection of fluorescence signals specifically associated with the proper genotypes in a mixture of amplified nontarget DNA molecules without DNA sizing. The measurement approach does not require complex, follow-up mathematical analysis and is applicable to any single nucleotide polymorphisms. The simple immunogenetic model showed how the approach works to reveal specific DNA target by preventing detection of nontarget DNA. Under those experimental conditions, a new ultrasensitive, and specific method for clinical immunologists is born.     

Relaxation times of choline, creatine and N-acetyl aspartate in human cerebral white matter at 1.5 T

Several studies have investigated the T1 and T2 relaxation time of choline, creatine and N-acetyl aspartate in cerebral white matter in normal human subjects. However, these studies demonstrate a large variation in T1 and T2 values. In the present study, relaxation times of choline, creatine and N-acetyl aspartate were determined in cerebral white matter in 15 control subjects (age 21 +/- 2 y, mean +/- SD) at 1.5 T. Using PRESS, seven or eight data points were obtained to fit the T1 and T2 relaxation curves to, respectively. The mean voxel size was 14 cm3. The T1 relaxation times of choline, creatine and N-acetyl aspartate were 1091 +/- 132 (mean +/- SD), 1363 +/- 137 and 1276 +/- 132 ms. The T2 relaxation times were 352 +/- 52, 219 +/- 29 and 336 +/- 46 ms, respectively.     

Self‐Assembly of Dendritic Macromolecules Based on the Ionic Interaction of Linear Chain Polyelectrolyte Cores with Oppositely Charged Focal Ionogenic Groups of Dendrons

Summary: A new principle for the design of dendritic macromolecules – the ionic binding of linear chain polyelectrolyte with oppositely charged focal ionogenic groups of dendrons – has been developed. The majority of the dendritic ionic complexes (DICs) are prepared with poly(styrenesulfonic acid) (PSS) as a polymeric core and L ‐aspartic acid dendrons of different generations. Two series of DICs were prepared using PSS and aspartic dendrons bearing terminal (located at the external periphery) methoxycarbonyl and hexyloxycarbonyl groups (C1‐n and C6‐n respectively where n is the generation number). Ionic binding of about 100% was found for dendrons of Generation 1–3. The solubility of the DICs was examined and the DICs prepared were studied by IR spectroscopy, ¹H NMR and viscometry.

Dendritic ionic complexes prepared using poly(styrenesulfonic acid) acid and aspartic dendrons bearing terminal methoxycarbonyl and hexyloxycarbonyl groups.     

Increased seminal plasma lead levels adversely affect the fertility potential of sperm in IVF

BACKGROUND: Lead remains in high levels in the environment and is known to reduce fertility in animal models, but a direct link between lead exposures and human infertility has not yet been established. METHODS: In a prospective, double-blind study of the metal ion levels and sperm function, semen was obtained from partners of 140 consecutive women undergoing their first IVF cycle. Lead in seminal plasma was determined by atomic absorption spectroscopy. Motile sperm populations were assessed for surface receptors for mannose binding, and the ability to undergo premature ('spontaneous'), and free mannose-induced acrosome reactions. Fertile donor (n = 9) sperm were exposed to exogenous lead during capacitating incubations and then assessed for mannose receptor expression and acrosome loss. RESULTS: Lead levels were negatively correlated with IVF rates. Lead levels were negatively correlated to two of the three sperm function biomarkers (mannose receptors, mannose-induced acrosome reactions). Lead levels positively correlated with the spontaneous acrosome reaction. These findings were mimicked by in-vitro exposure of fertile donor sperm to lead. CONCLUSIONS: Multiple sperm parameters are affected as lead levels rise. Increased lead levels may contribute to the production of unexplained male infertility.