A paradigm of experimentally induced mild hyperthyroidism: effects on nitrogen balance, body composition, and energy expenditure in healthy young men

OBJECTIVE: To conceptualize, with fine needle aspiration cytology (FNAC), the early cellular events occurring in and around fresh autogenous and allogenic bone grafts during the first 40 postimplantation days. STUDY DESIGN: Forty-eight cases of bone grafts were studied by FNAC at serial intervals of 10, 20, 30 and 40 postimplantation days. Twenty patients were recipients of autogenous grafts, 16 received 0.6N HCI partially decalcified allogenic bone implants, and 4 received combined autogenous and allogenic bone grafts (included in the allograft group). There were eight control cases of closed fracture shaft femur, which were managed conservatively. RESULTS: The initial cellular responses in autogenous grafts, allografts and controls appear to be a part of the nonspecific reparative process followed by a more specific phase, with a steady increase in relative lymphocyte count from the 20th day onwards. Osteogenesis, as judged by osteoblasts and osteoclasts, was also comparable. CONCLUSION: Partially decalcified allografts appear to be a good substitute for autogenous bone grafts in clinical practice when adequate autogenous material is not available. FNAC is a good technique for studying bone graft responses without interfering with graft uptake. It is helpful in the early detection of subclinical infection or any other pathology at the graft site.     

Herpes simplex virus seropositivity and reactivation at delivery among pregnant women infected with human immunodeficiency virus-1

OBJECTIVE: Our purpose was to determine whether pregnant women infected with human immunodeficiency virus-1 have an increased risk of herpes simplex virus-2 seropositivity and herpes simplex virus reactivation at delivery. STUDY DESIGN: Sixty women infected with human immunodeficiency virus and 8408 other patients who were delivered at the University of Washington between 1989 and 1995 had herpes simplex virus serologic determinations at delivery. Genital herpes simplex virus cultures were obtained for 48 (80%) of the human immunodeficiency virus-infected women and 5567 (66%) of the controls. Logistic regression was used to adjust for possible confounding factors. RESULTS: Forty-five (75%) of human immunodeficiency virus-infected women and 2709 (32%) controls were seropositive for herpes simplex virus-2 (p < 0.0001). Eight percent of human immunodeficiency virus-infected women and 2% of controls had herpes simplex virus reactivation in labor (p < 0.05). CONCLUSIONS: Infection with herpes simplex virus-2 is common among pregnant women infected with human immunodeficiency virus. Herpes simplex virus reactivation complicates labor in this group more often than in other obstetric patients. The role of herpes simplex virus in perinatal human immunodeficiency virus transmission warrants further study.     

Improving mass spectrometric sequencing of arginine-containing peptides by derivatization with acetylacetone

Modification of arginine residues in bradykinin, [1-5]-bradykinin, splenopentin and two synthetic pentapeptides with acetylacetone (pentane-2,4-dione) significantly increases the relative abundance of sequence-specific fragment ions produced by matrix-assisted laser desorption/ionization (MALDI). The fragmentation efficiency as measured by post-source decay in a reflectron time-of-flight mass spectrometer increases by a factor of 2-3.5. Peptide bonds adjacent to modified residues are more susceptible to cleavage than in the non-derivatized peptide ions. The increased lability of these bonds gives rise to more complete sequence information. In addition, the relative abundances of sequence-specific fragment ions are enhanced. This strategy makes it possible to obtain valuable structural information from arginine-containing peptides that otherwise do not fragment well.     

Progression of interproximal caries in the primary dentition

In the patients with invasion to the aortic window, we performed operation via median sternotomy combined with anteroaxillar thoracotomy. In such patients with T4 invasion, conventional pneumonectomy could not be performed because of the extensive invasion near the main pulmonary artery trunk. In these patients in this study, complete resection of the involved pulmonary artery could be performed using a vascular clamp without CP bypass. Operative technique was as follows: first, the pericardium was opened and taping of the aorta was applied. When the uninvolved part of the intrapericardial pulmonary artery was long enough to cut, we could use a stapling device, but the stapling device could not be used in many cases because the length of the uninvolved segment was too short to cut the left pulmonary artery. In order to carry out complete resection, it was necessary to clamp the central part of the main pulmonary artery diagonally from the left lower side to the right upper side. The pulmonary arterial stump was closed with continuous 4-0 monofilament mattress and over and over suture. We recommend an aggressive surgical approach for the tumor with invasion to the aortic window, because the prognosis is dismal in nonresected locally advanced lung cancer.     

Tumor necrosis factor-alpha and interleukin-1 induce activation of MAP kinase and SAP kinase in human neuroma fibroblasts

Two cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1), which are released by macrophages during the early inflammatory phase of nerve injury, are known to induce activation of mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK), which locate at different signal transduction pathways and are involved in cell cycle G0/G1 transition and cellular proliferation in human fibroblasts. Activation of these two protein kinases by the cytokines may stimulate fibroblast proliferation in damaged nerves and thereby play a role in the formation of a neuroma, a disorganized mass of tissue that interferes with neural regeneration and repair. To investigate the possibility that this mechanism is operative in neuroma formation, we used cultured, serum-starved fibroblasts from surgically removed human neuromas stimulated with TNF-alpha and/or IL-1 alpha and IL-1 beta, and measured the activation of MAPK and SAPK using myelin basic protein (MBP) and human c-Jun (1-169) glutathione S-agarose transferase (GST) fusion protein as substrates. For comparison, neuroma fibroblast cultures were also stimulated with phorbol 12-myristate 13-acetate (PMA) and platelet-derived growth factor-AB (PDGF-AB), a potent activator for MAPK. TNF-alpha and both forms of IL-1 produced a rapid activation of MAPK, with a peak at 15 min for TNF-alpha stimulation, and a peak at 30 min for IL-1 stimulation. TNF-alpha combined with either IL-1 alpha or IL-1 beta produced a synergistic effect on the activation of MAPK. The increases in MAPK induced by TNF-alpha and IL-1 were similar to the increases induced by PMA and PDGF-AB. To confirm the presence of MAPK, immunoprecipitation and immunoblotting were carried out on experimental and control lysates. TNF-alpha and IL-1 also increased activation of SAPK, but to a lesser extent than MAPK. PMA and PDGF-AB were also much less effective in stimulating activation of SAPK. Our findings indicate that TNF-alpha and IL-1 activate parallel signal transduction pathways in human neuroma fibroblasts, and that they are relatively stronger activators of MAPK than of SAPK. Previous studies have convincingly demonstrated that MAPK and SAPK are involved in human fibroblast proliferation. The results of our study suggest that TNF-alpha and IL-1 may play a role in frustrating functional nerve regeneration after injury by stimulating these two kinases, which, in turn, leads to fibroblast proliferation and formation of neuromas.     

Stereochemical course of enzymatic enolpyruvyl transfer and catalytic conformation of the active site revealed by the crystal structure of the fluorinated analogue of the reaction tetrahedral intermediate bound to the active site of the C115A mutant of MurA

MurA (UDP-GlcNAc enolpyruvyl transferase), the first enzyme in bacterial peptidoglycan biosynthesis, catalyzes the enolpyruvyl transfer from phosphoenolpyruvate (PEP) to the 3'-OH of UDP-GlcNAc by an addition-elimination mechanism that proceeds through a tetrahedral ketal intermediate. The crystal structure of the Cys115-to-Ala (C115A) mutant of Escherichia coli MurA complexed with a fluoro analogue of the tetrahedral intermediate revealed the absolute configuration of the adduct and the stereochemical course of the reaction. The fluorinated adduct was generated in a preincubation of wild-type MurA with (Z)-3-fluorophosphoenolpyruvate (FPEP) and UDP-GlcNAc and purified after enzyme denaturation. The fluorine substituent stabilizes the tetrahedral intermediate toward decomposition by a factor of 10(4)-10(6), facilitating manipulation of the adduct. The C115A mutant of MurA was utilized to avoid the microheterogeneity that arises in the wild-type MurA from the attack of Cys115 on C-2 of FPEP in competition with the formation of the fluorinated adduct. The crystal structure of the complex was determined to 2.8 A resolution, and the absolute configuration at C-2 of the adduct was found to be 2R. Thus, addition of the 3'-OH of UDP-GlcNAc is to the 2-si face of FPEP, corresponding to the 2-re face of PEP. Given the previous observation that, in D2O, the addition of D+ to C-3 of PEP proceeds from the 2-si face [Kim, D. H., Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 6380-6381], the addition across the double bond of PEP is anti. Also, because the overall stereochemical course has been shown to be either anti/syn or syn/anti [Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 7329-7337], it now follows that the stereochemistry of elimination of H+ from C-3 and Pi from C-2 of the tetrahedral intermediate of the reaction is syn.     

Limitations to exercise- and maximal insulin-stimulated muscle glucose uptake

The hypothesis of this investigation was that insulin and muscle contraction, by increasing the rate of skeletal muscle glucose transport, would bias control so that glucose delivery to the sarcolemma (and t tubule) and phosphorylation of glucose intracellularly would exert more influence over glucose uptake. Because of the substantial increases in blood flow (and hence glucose delivery) that accompany exercise, we predicted that glucose phosphorylation would become more rate determining during exercise. The transsarcolemmal glucose gradient (TSGG; the glucose concentration difference across the membrane) is inversely related to the degree to which glucose transport determines the rate of glucose uptake. The TSGG was determined by using isotopic methods in conscious rats during euglycemic hyperinsulinemia [Ins; 20 mU/(kg. min); n = 7], during treadmill exercise (Ex, n = 6), and in sedentary, saline-infused rats (Bas, n = 13). Rats received primed, constant intravenous infusions of trace 3-O-[3H]methyl-D-glucose and [U-14C]mannitol. Then 2-deoxy-[3H]glucose was infused for the calculation of a glucose metabolic index (Rg). At the end of experiments, rats were anesthetized, and soleus muscles were excised. Total soleus glucose concentration and the steady-state ratio of intracellular to extracellular 3-O-[3H]methyl-D-glucose (which distributes on the basis of the TSGG) were used to calculate ranges of possible glucose concentrations ([G]) at the inner and outer sarcolemmal surfaces ([G]im and [G]om, respectively). Soleus Rg was increased in Ins and further increased in Ex. In Ins, total soleus glucose, [G]om, and the TSGG were decreased compared with Bas, while [G]im remained near 0. In Ex, total soleus glucose and [G]im were increased compared with Bas, and there was not a decrease in [G]om as was observed in Ins. In addition, accumulation of intracellular free 2-deoxy-[3H]glucose occurred in soleus in both Ex and Ins. Taken together, these data indicate that, in Ex, glucose phosphorylation becomes an important limitation to soleus glucose uptake. In Ins, both glucose delivery and glucose phosphorylation influence the rate of soleus glucose uptake more than under basal conditions.