Arachidonic acid metabolites and enzyme transcripts in asthma are altered by cigarette smoking.

BACKGROUND: Arachidonic acid metabolites are implicated in the pathogenesis of asthma although only limited information is available on the impact of current smoking history on these metabolites. The aim of the study was to examine the effect of smoking status on urinary, sputum, and plasma eicosanoid concentrations and relevant enzyme transcripts in asthma. METHODS: In 108 smokers and never smokers with asthma and 45 healthy controls [smokers and never smokers], we measured urinary tetranor prostaglandin (PG)D2 (PGDM) and leukotriene (LT)E4 , induced sputum fluid LTB4 , LTE4 , PGD2 , and PGE2 , plasma secretory phospholipase A2 (sPLA2 ), and 11ß prostaglandin F2α (11ßPGF2α ), and, in a subgroup with severe asthma, airway leukocyte and epithelial cell mRNA expression levels of arachidonic acid metabolic enzymes. RESULTS: Smokers with asthma had higher urinary LTE4 ; 83 (59, 130) vs 59 (40, 90) pg/mg creatinine, P = 0.008, and PGDM; 60 (35, 100) vs 41 (28, 59) ng/mg creatinine, P = 0.012 concentrations, respectively, and lower sputum PGE2 concentrations 80 (46, 157) vs 192 (91, 301) pg/ml, P = 0.001 than never smokers with asthma. Sputum LTB4 (P = 0.013), and plasma 11ßPGF2α (P = 0.032), concentrations, respectively, were increased in smokers with asthma compared with healthy smokers. Asthma-specific and smoking-related increases (>1.5-fold expression) in arachidonate 15-lipoxygenase and gamma-glutamyltransferase transcripts were demonstrated. CONCLUSIONS: Several arachidonic acid metabolites and enzyme transcripts involving both lipoxygenase and cyclooxygenase pathways are increased in smokers with asthma and differ from never smokers with asthma. Possibly targeting specific lipoxygenase and cyclooxygenase pathways that are activated by asthma and cigarette smoking may optimize therapeutic responses.

Apopain/CPP32 cleaves proteins that are essential for cellular repair: a fundamental principle of apoptotic death.

Proteolysis mediated by the interleukin 1 beta-converting enzyme (ICE) homologues is an important mechanism of the apoptotic process. The ICE homologue apopain/CPP-32/Yama (subsequently referred to as apopain) cleaves poly(ADP-ribose)polymerase (PARP) early during apoptosis. Additional apoptosis-specific protein cleavages have been observed in which the direct involvement of ICE-like proteases has been postulated. These substrates include the 70-kD protein component of the U1-ribonucleoprotein (U1-70kD), and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). The present studies demonstrate that U1-70kD and DNA-PKcs are excellent substrates for apopain, with cleavage occurring at sites that are highly similar to the cleavage site within PARP. The fragments generated from isolated protein substrates by apopain are identical to those observed in intact apoptotic cells, in apoptotic cell extracts, and in normal cell extracts to which apopain has been added. Like PARP, cleavage of these substrates in apoptotic cell extracts is abolished by nanomolar concentrations of Ac-DEVD-CHO and micromolar amounts of Ac-YVAD-CHO, confirming the involvement of apopain or an apopain-like activity. We propose that a central function of apopain or similar homologues in apoptosis is the cleavage of nuclear repair proteins, thereby abolishing their critical homeostatic functions.

The interleukin-1 beta-converting enzyme (ICE) is localized on the external cell surface membranes and in the cytoplasmic ground substance of human monocytes by immuno-electron microscopy.

Interleukin-1 beta (IL-1 beta)-converting enzyme (ICE) is a novel cysteine protease that cleaves the 31-kD inactive cytoplasmic IL-1 beta precursor into active extracellular 17-kD IL-1 beta. The ICE gene product is a 45-kD proenzyme that requires proteolytic processing to activate ICE. Active ICE is a heterodimer consisting of equal amounts of p20 and p10 subunits. Generation of active ICE is affected by the removal of an 11-kD NH2-terminal precursor domain (p11) and an internal 19-amino acid sequence that separates the 20- and 10-kD subunits. Immuno-electron microscopy was performed on human monocytes with immunoglobulins recognizing the active (p20) or precursor (p11) domains of ICE. Elutriated monocytes were stimulated with 50 pM lipopolysaccharide followed by heat-killed Staphylococcus aureus under conditions that induce maximal rates of IL-1 beta secretion. Ultrathin cryosections were cut from fixed frozen pellets of these monocytes and were immunogold labeled with either antibody. Active and precursor domain ICE epitopes were localized in the cytoplasmic ground substance, but they were not detected within the endoplasmic reticulum, the Golgi apparatus, and secretory granules of activated or inactive monocytes. Importantly, numerous ICE p20 epitopes were also observed on the extracellular surfaces of the cell membrane, and were concentrated on the microvilli. Very similar patterns of ICE localization were obtained with unstimulated blood monocytes. In contrast, ICE p11 epitopes were not detected on the surfaces of these monocytes. Likewise, labeling of fixed ultrathin cryosections of monocytes with a biotinylated irreversible ICE inhibitor [Ac-Tyr-Val-Lys(biotin)-Asp-(acyloxy)-methyl-ketone] showed that the compound localized on the outer cell surface as well, and to a lesser extent, within the cytoplasmic ground substance. Furthermore, antipeptide antibodies specific for either the mature or precursor domains of IL-1 beta were both localized upon the cell membrane after stimulation of IL-1 beta secretion. Lipopolysaccaride-primed monocytes that synthesized, but did not secrete IL-1 beta, exhibited only cytoplasmic staining. The data suggests that mature IL-1 beta is generated via cleavage of the 31-kD inactive cytoplasmic IL-1 beta precursor by ICE after association with the plasma membrane during secretion.

Inhibition of vesicular stomatitis virus infection by spike glycoprotein. Evidence for an intracellular, G protein-requiring step.

In an assay measuring virus-directed RNA synthesis, infection of BHK cells by a standard test dose of vesicular stomatitis virus (VSV) was inhibited by ultraviolet light-irradiated wt VSV and by ts 045, one of a number of thermolabile, temperature-sensitive G protein mutants of VSV. After heat treatment for 1 h at 45 degrees C, the thermolabile mutants were no longer able to inhibit the VSV infection. In contrast, the thermolabile M protein mutant ts G31 and the nonthermolabile G protein mutant ts 044 could still inhibit the test VSV dose. Thus, the presence of G protein in its native conformation was necessary for inhibition of infection. There was little difference in the binding to cells or the internalization to a trypsin-resistant state of ts 045 or wt VSV before and after heat treatment, and there was no evidence of specific saturable receptors on the cell surface. None of the irradiated virions at concentrations that gave maximal inhibition of infection could prevent binding of infectious VSV to, or internalization by, BHK cells. The G protein-specific inhibition, therefore, did not occur at the cell surface but must have occurred at some intracellular site, which has been suggested to be the lysome. The lysosomal inhibitor chloroquine, when added with the infecting virus, completely inhibited VSV infection at all multiplicities of infection tested, and it gave 50% inhibition when added to 1.5 h after infection. The possible importance of the lysosome in the intracellular pathway of infection is discussed.

Reconstituted G protein-lipid vesicles from vesicular stomatitis virus and their inhibition of VSV infection.

The single glycoprotein (G) of vesiclar stomatitis virus (VSV) was isolated in nearly quantitative yield by extraction of the purified virions with 0.05 M octyl-beta-D- glucoside (OG) in 0.01 M sodium phosphate, pH 8.0. The extract contained essentially all of the viral phospholipids and glycolipids, and was free of other essentially all of the viral phospholipids and glycolipids, and was free of other viral proteins. Dialysis to remove OG resulted in the formation of G protein-viral lipid vesicles having a lipid-G protein ratio similar to that of the intact virions. The vesicles were 250-1,000 A in diameter, with a "fuzzy" external layer also similar to that of intact virions. The vesicles were predominantly unilamellar and sealed, with both phosphatidyl ethanolamine and gangliosides symmetrically distributed in the bilayer. G protein was asymmetrically oriented, with about 80 percent accessible to exogenous protease. Addition of soybean phospholipid to the viral extract before dialysis resulted in vesicles that incorporated viral proteins and lipids quantitatively, but that were markedly decreased in buoyant density. The G neutralized protein-lipid vesicles were effective in eliciting specific anti-G antibodies that neutralized viral infectivity. Competitive radioimmunoassay showed that both reconstituted vesicles and a soluble form of G protein (Gs) were indistinguishable from purified VSV in their antibody binding properties. Addition of G protein-lipid vesicles of BHK-21 cells before, or simultaneously with, infection by VSV inhibited viral infectivity, as measured by two independent techniques (viral RNA production in the presence of actinomycin D and a neutral red assay of cell viability). The total inhibitory activity of G protein in the vesicular form was, however, less than 5 percent of that found for intact virus particles that have been inactivated by ultraviolet light irradiation. Gs was inactive as an inhibitor as determined by the RNA production assay.

Cell killing by lysosomotropic detergents.

We have studied the mechanism by which lysosomotropic detergents kill baby hamster kidney cells. Lysosomotropic detergents are lysosomotropic amines (compounds with pK between 5 and 9, such as imidazole or morpholine) containing straight-chain hydrocarbon "tails" of 9-14 carbon atoms (Firestone, R. A., J. M. Pisano, and R. J. Bonney. 1979, J. Med. Chem., 22:1130-1133). Using lucifer yellow CH as a specific fluorescent label for lysosomes, it was shown by light microscopy that N-dodecyl (C12)-imidazole acted rapidly to damage lysosomes, causing leakage of dye into the cytoplasm. This was followed at later times by vacuolization, blebbing of the plasma membrane, cell rounding, and cell death. 3H-labeled C12-imidazole rapidly diffused into cells where much of it was trapped in lysosomes as shown by its co-migration with lysosomes in Percoll gradients. Cells preincubated with C12-imidazole released it slowly into C12-imidazole-free media, permitting the cells to be killed by the preincubation dose. Cell killing by the lysosomotropic detergents exhibited strongly sigmoidal dose-response curves. The sensitivity of baby hamster kidney cells to killing by C12-imidazole was density dependent, the cells being most sensitive at lowest cell densities, and relatively resistant at confluence. The amount of 3H-C12-imidazole taken up by the cells was also density dependent, with highest specific uptake occurring at the lowest cell density. A rise in lysosomal pH, measured in fluoresceinated dextran-labeled cells, commenced immediately upon addition of C12-imidazole to cells, and continued for over an hour. This was followed after a lag of 1-2 h by inhibition of protein and RNA synthesis and by lactate dehydrogenase release. Ionophores or lysosomotropic amines, such as methylamine, that raise intralysosomal pH provided substantial protection of the cells from killing by lysosomotropic detergents. These findings provide strong support for the idea that lysosomotropic detergents kill cells by disrupting lysosomes from within.

Screening for Vitamin D Deficiency in Black Americans: Comparison of Total, Free, Bioavailable 25 Hydroxy Vitamin D Levels with Parathyroid Hormone Levels and Bone Mineral Density.

OBJECTIVES: There is debate surrounding the adequacy of total and free 25 hydroxy vitamin D [25(OH)D] levels in black Americans who have inherently high bone mineral density [BMD] and low serum concentration of vitamin D binding proteins [VDBP]. DESIGN: Retrospective analysis of serum samples and BMD analyses from the African American Health Study [AAHS] cohort. SETTING: The AAHS is a population-based longitudinal study initiated to examine issues of disability and frailty among urban-dwelling black Americans in the city of Saint Louis, Missouri. PARTICIPANTS: 122 men and 206 women, age 60.2 ± 4.3 years. INTERVENTION: Retrospective analysis. MEASUREMENTS: Total 25(OH)D, VDBP, PTH, and BMD of the lumbar spine and hip by dual energy x-ray photometry (DXA). Free and bioavailable vitamin D levels were calculated using serum concentrations and affinity constants for the VDBP (Gc1F and Gc1S) phenotypes. RESULTS: Serum total 25(OH)D levels were 14.6 ± 8.9 ng/mL (36 ± 22 nmol/L). Vitamin D insufficiency was estimated by compensatory elevations of PTH above the normal range (> 65 pg/mL). PTH levels were within the normal reference range in > 95% of the samples at total 25(OH)D levels ≥ 20 ng/mL (≥50 nmol/L). There was no difference in the correlation of the reciprocal relationship of vitamin D vs parathyroid hormone between the VDBP phenotypes. Receiver operating characteristic curve analyses indicated that serum total 25(OH)D discriminated sufficiency from insufficiency at least as well as the calculated levels of the free and bioavailable vitamin D. Very low levels of total 25(OH)D (≤ 8 ng/mL, ≤20 nmol/L) were associated with decreased BMD (p=0.02), but higher levels of 25(OH)D did not show statistical differences in BMD. CONCLUSION: Total 25(OH)D levels of ≤ 8ng/mL (≤20 nmol/L) are associated with clinically significant changes in BMD, whereas total 25(OH)D levels ≥ 20 ng/mL (≥50 nmol/L) suppressed PTH and were not associated with deficiencies in BMD. Lower levels of 25(OH)D may be acceptable for bone health in black than in white Americans.

Loss of strain specificity of the TA3-St subline: evidence for the role of epiglycanin in mouse allogeneic tumor growth.

We described the in vivo conversion of the strain-specific ascites murine mammary adenocarcinoma subline TA3-St to a new ascites subline designated TA3-MM. This conversion occurred during passage in a syngeneic A/HeHa mouse infected with pneumonia-producing microorganisms. The mode number of chromosomes of the TA3-MM cell (82) was greater than that of the parental TA3-St cell (69) or the other non-strain-specific subline TA3-Ha (42). The TA3-MM subline could grow in and kill mice of various allogeneic strains. In addition, the TA3-MM cell possessed numerous receptors for the lectin of Vicia graminea seeds, which were hardly detectable at the surface of the parent TA3-St subline but were present in abundance at the cell surface of the non-strain-specific subline TA3-Ha. These lectin receptors of the TA3-Ha cell were previously demonstrated to be present in a unique high-molecular-weight endogenous cell surface glycoprotein termed epiglycanin. The V. gramines lectin receptors on the new TA3-MM subline also were present on an epiglycanin-like molecule. This finding provides further evidence for the hypothesis that allogeneic growth in the TA3 system is a direct result of these membrane glycoproteins.

Reversible loss in suspension culture of a major cell-surface glycoprotein of the TA3-Ha mouse tumor.

A major cell-surface glycoprotein of the TA3-Ha ascites mammary adenocarcinoma diminished during transfer from ascites growth to cell growth in suspension culture. A sensitive, hemagglutination-inhibition assay that used a lectin from Vicia graminea seeds indicated approximately a 50% loss after 7-10 days of culture and a 90% loss after 2 months. These findings were corroborated by carbohydrate and amino acid analysis with gas-liquid chromatography of trypsin glycopeptides released from the cell surface. Repassage of the cultured cells in vivo caused the reappearance of the surface glycoprotein.

Isolation and partial characterization of an epiglycanin-like glycoprotein from a new non-strain-specific subline of TA3 murine mammary adenocarcinoma.

A new non-strain-specific ascites subline of the TA3 mammary adenocarcinoma TA3-MM, which arose in vivo from the strain-specific TA3-St subline during an acute respiratory illness of the syngeneic mouse strain A/HeHa hosts, possessed at its surface a glycoprotein not found on the parent TA3-St cell. This glycoprotein, termed TA3-MM epiglycanin, was characterized by a high molecular weight (500,000), by potent inhibition of hemagglutination by the Vicia gramines lectin, and by carbohydrate and amino acid compositions nearly identical to those of the glycoprotein epiglycanin present at the surface of the allotransplantable TA3-Ha ascites cell. By electron microscopic examination, TA3-MM epiglycanin appeared as long extended rods with widths (2.5 nm) and lengths (450--500 nm) similar to those of TA3-Ha epiglycanin. Incubation of each of two sublines of the TA3-MM ascites cell, TA3-MM/1 and TA3-MM/2, with a modified trypsin followed by column chromatography produced approximately 1.0- and 0.2-fold as much epiglycanin-like material, respectively, as was obtained from the TA3--a ascites cell. Continuous growth of the TA3-MM cell in suspension culture resulted in an almost complete disappearance of epiglycanin in a manner demonstrated earlier for the TA3-Ha cell grown under similar conditions. Allotransplantability in the TA3-MM cell may be due, at least in part, to masking a histocompatibility antigens by epiglycanin-like molecules.

Frailty, Diabetes, and Mortality in Middle-Aged African Americans.

BACKGROUND: Older adult frail diabetics have high mortality risk, but data are limited regarding frail late middle-aged diabetics, especially for African-Americans. The aim of this study is to examine the association of diabetes with health outcomes and frailty in the African American Health (AAH) study. METHODS: AAH is a population-based longitudinal cohort study. Participants were African Americans (N=998) ages 49 to 65 years at baseline. Cross-sectional comparisons for diabetes included disability, function, physical performance, cytokines, and frailty. Frailty measures included the International Academy of Nutrition and Aging [FRAIL] frailty scale, Study of Osteoporotic Fractures [SOF] frailty scale, Cardiovascular Health Study [CHS] frailty scale, and Frailty Index [FI]). Longitudinal associations for diabetes included new ADLs ≥ 1 and mortality at 9-year follow-up. RESULTS: Diabetics were more likely to be frail using any of the 4 frailty scales than were non-diabetics. Frail diabetics, compared to nonfrail diabetics, reported significantly increased falls in last 1 year, higher IADLs and higher LBFLs. They demonstrated worse performance on the SPPB, one-leg stand, and grip strength; and higher Tumor Necrosis Factor receptors (sTNFR1 and sTNFR2). Mortality and 1 or more new ADLs also were increased among frail compared to nonfrail diabetics when followed for 9 years. CONCLUSIONS: Frailty in middle-aged African American persons with diabetes is associated with having more disability and functional limitations, worse physical performance, and higher cytokines (sTNFR1 and sTNFR2 only). Middle-aged African Americans with diabetes have an increased risk of mortality and frail diabetics have an even higher risk of death, compared to nonfrail diabetics.

Fruit and Vegetable Intake and Physical Activity as Predictors of Disability Risk Factors in African-American Middle-Aged Individuals.

OBJECTIVE: To investigate fruit and vegetable intake (FVI) and different dimensions of physical activity (PA) as predictors of change in disabilities and other known precursors of progressive disability in a population-based sample of African Americans. DESIGN: Longitudinal investigation of the independent associations of reported FVI and PA with six-year changes in disabilities and other known precursors of progressive disability. SETTING: Longitudinal study of a population-representative cohort of late middle-aged African Americans. PARTICIPANTS: 432 cohort participants with complete information on all measures. Measurements and Analytic Approach: During wave 8 (2008), FVI was measured using 2005 Behavioral Risk Factor Surveillance System questions and PA dimensions using the Yale Physical Activity Survey (YPAS). Disability measures included basic activities of daily living (ADLs) and instrumental ADLs (IADLs); other precursors included measured gait speed, grip strength, and short physical performance battery (SPPB) and reported lower body functional limitations (LBFLs) and FRAIL scale; these were measured at wave 4 (2004) and wave 10 (2010). Residual-change score linear regression was used to identify FVI and PA factors that were independently associated with six-year changes in disability and other precursors. RESULTS: The study cohort was less active than the YPAS-development group. Longitudinally, leisurely walking was independently associated with better ADL, IADL, grip strength, SPPB, LBFL, and frailty outcomes; standing with better IADL and SPPB; intake of vegetables other than carrots, salads, or potatoes with better grip strength and frailty; and fruit juice intake with worse grip strength and frailty. CONCLUSIONS: In this relatively inactive cohort, leisurely walking was associated with multiple beneficial outcomes. Benefits were also seen with vegetables other than potato intake, and fruit juice intake was associated with detrimental effects. This study highlights the importance of finding strategies to help this population increase PA (especially leisurely walking) and intake of whole fruits and vegetables.

Cocaine self-administration and reinstatement in female rats selectively bred for high and low voluntary running.

BACKGROUND: Previous research has found that rats behaviorally screened for high (vs. low) wheel running were more vulnerable to cocaine abuse. To assess the extent to which a genetic component is involved in this drug-abuse vulnerability, rats selectively bred for high or low voluntary running (HVR or LVR, respectively) were examined for differences in cocaine seeking in the present study. METHODS: Female rats were trained to lever press for food and then were assessed for differences in acquisition of cocaine (0.4mg/kg; i.v.) self-administration across 10 sessions. Once acquired, rats self-administered cocaine for a 14-day maintenance phase, followed by a 14-day extinction phase when cocaine was no longer available. Subsequently, reinstatement of cocaine seeking was examined with priming injections of cocaine (5, 10 & 15mg/kg), caffeine (30mg/kg), yohimbine (2.5mg/kg) and cocaine-paired cues. RESULTS: A greater percentage of LVR rats met the acquisition criteria for cocaine self-administration and in fewer sessions than HVR rats. No differences in responding for cocaine were observed between phenotypes during maintenance. However, during extinction LVR rats initially responded at higher rates and persisted in cocaine seeking for a greater number of sessions. No phenotype differences were observed following drug and cue-primed reinstatement of cocaine seeking. CONCLUSIONS: In general, LVR rats were more sensitive to the reinforcing effects of cocaine than HVR rats during periods of transition into and out of cocaine self-administration. Thus, LVR rats sometimes showed a greater vulnerability cocaine seeking than HVR rats.

Actin cleavage by CPP-32/apopain during the development of apoptosis.

Interleukin-1beta-converting enzyme (ICE)/ced-3 family proteases play key roles in apoptosis. However, cellular substrates for ICE family proteases involved in apoptosis are not well understood. We previously showed that actin is cleaved in vitro by an ICE family protease, distinct from ICE itself, which is activated during VP-16-induced apoptosis. In this report, we demonstrate that the actin-cleaving ICE-family protease in the apoptotic cell extract is the activated CPP-32/apopain. CPP-32 effectively cleaves actin protein to 15 kDa and 31 kDa fragments. Studies with an antibody raised against Gly-Gln-Val-Ile-Thr peptide, the N-terminal sequence of the cleaved 15 kDa actin fragment, showed that actin is also cleaved in vivo during the development of apoptosis. Moreover, Benzyloxycarbonyl-Glu-Val-Asp-CH2OC(O)-2,6,-dichlorobenzene (Z-EVD-CH2-DCB), a selective inhibitor of CPP-32(-like) protease, efficiently inhibited the cleavage of actin and the apoptosis of VP-16-treated U937 cells. Our present results indicate that actin is the substrate of CPP-32/apopain(-like) protease both in vitro and in vivo and suggest the role of actin in the control of cell growth and apoptosis.

Cost-effectiveness of thyroid function tests.

The cost-effectiveness of thyroid function tests (serum thyroxine concentration, triiodothyronine [T3]-resin uptake, free thyroxine index, serum T3, and serum thyrotropin concentration) was assessed in 135 ambulatory patients suspected of hypothyroidism or hyperthyroidism who did not have a history of thyroid disease requiring medication or thyroid surgery within the preceding two years. Of patients with five or more signs and symptoms compatible with thyroid dysfunction, 50.0% had biochemical abnormalities substantiating hypothyroidism or hyperthyroidism, while only 1.5% of patients with fewer than two signs and symptoms had either disease. The cost of thyroid function tests was twice as much per patient evaluated by residents as for those evaluated by faculty physicians. These results suggest that interventions to reduce the number and type of tests in patients without multiple signs and symptoms of thyroid disease could improve the cost-effective use of these tests.

Time and financial analysis of an academic general internal medicine unit.

A detailed time and financial analysis of an academic general internal medicine unit was used to examine financial, educational, and research issues. The residents' outpatient service possessed the greatest patient visit and revenue capacity, but revenue recovery was limited by low productivity and collection rate. Outpatient revenue production could be improved but would still be less than current inpatient revenues. Full financial self-sufficiency would require improvements in several aspects of revenue production and would severely limit faculty time for teaching, personal study, and research. Indirect financial contributions to the medical center through hospitalizations and referrals dwarfed direct revenues. Several strategies for academic general internal medicine units to achieve financial and academic survival are possible, each with its own set of trade-offs. Because local circumstances differ, strategies and outcomes are likely to vary among units.

The effect of prescribed daily dose frequency on patient medication compliance.

The objective of this study was to determine the relationship between prescribed daily dose frequency and patient medication compliance. The medication compliance of 105 patients receiving antihypertensive medications was monitored by analyzing data obtained from special pill containers that electronically record the date and time of medication removal. Inaccurate compliance estimates derived using the simple pill count method were thereby avoided. Compliance was defined as the percent of days during which the prescribed number of doses were removed. Compliance improved from 59.0% on a three-time daily regimen to 83.6% on a once-daily regimen. Thus, compliance improves dramatically as prescribed dose frequency decreases. Probably the single most important action that health care providers can take to improve compliance is to select medications that permit the lowest daily prescribed dose frequency.

Osteomyelitis caused by Mycobacterium haemophilum: successful therapy in two patients with AIDS.

OBJECTIVE: Difficulties involved in diagnosis and response to antimicrobial therapy are described in detail for two cases of biopsy-proven osteomyelitis caused by Mycobacterium haemophilum in AIDS patients. SETTING: Two large, private teaching hospitals in New York City, New York, USA. PATIENTS, PARTICIPANTS: A 31-year-old woman with previous diagnoses of candida esophagitis and peripheral neuropathy (patient 1), and a 37-year-old man with Kaposi's sarcoma (patient 2). INTERVENTIONS: One patient was treated with a combination of rifampin, ethambutol, clofazimine, and ciprofloxacin, while the other received rifampin, ciprofloxacin and doxycycline. Both patients also received a short course of intravenous amikacin. MAIN OUTCOME MEASURES: Disease activity was monitored clinically by observing resolution of skin ulcers, lymphadenopathy, and pain and swelling in areas affected by osteomyelitis. RESULTS: Both patients experienced complete resolution of signs and symptoms of M. haemophilum infection. Patient 1 was treated for 17 months and remains well after 10 months without therapy. Patient 2 shows no evidence of infection after 14 months of therapy. CONCLUSIONS: M. haemophilum infection must be considered in the differential diagnosis of osteomyelitis in AIDS patients, although specialized culture techniques are required to isolate and identify this pathogen. Excellent clinical response can be achieved with oral antimicrobial therapy.

Aging and bone metabolism in African American and Caucasian women.

There is limited information concerning bone mineral density (BMD) and its determinants across a wide spectrum of ages in African American females (AAF). Therefore, we have performed a cross-sectional study of 54 AAF and 39 Caucasian females (CF), aged 20-90 yr, to quantify femoral and lumbar bone mineral density, total body calcium, as well as the potential determinants of bone density. BMD decreased with age in all sites after age 40 yr in both racial groups. Bone density was greater in AAF than in CF, although there was considerable overlap between the two groups. There was no significant difference in the rate of age-related bone loss between the two groups. At the femoral neck, BMD was below the fracture threshold in 28% of the postmenopausal AAF compared to 47% of postmenopausal CF. L1-L4 BMD was below the fracture threshold in 8% of postmenopausal AAF and 11% of postmenopausal CF. Serum-25 hydroxyvitamin D (25OHD) was inversely related to age in both othnic groups and lower (P < 0.05) in premenopausal AAF than CF. Twenty-four percent of the AAF and 22% of the CF had serum 25OHD levels of 8 ng/L or less. Serum PTH was directly related to age (r = 0.43; P = 0.003 in AAF and r = 0.55; P = 0.002 in CF), and 25OHD was inversely related to age (r = -0.43; P = 0.003 in AAF and r = -0.65; P < 0.001 in CF). PTH was higher (P < 0.05) in postmenopausal AAF than in CF. Serum testosterone was greater in AAF than in CF (P < 0.05). Serum estradiol was greater in premenopausal AAF than in CF. Serum dehydroepiandrosterone sulfate was inversely related to age (r = 0.42; P = 0.004 in AAF and r = 0.51; P = 0.005 in CF). Serum osteocalcin was related to age in AAF (r = 0.47; P = 0.001), but not in CF. There was also a trend for an increase in urinary dipyridinoline (expressed per 100 mg urinary creatinine) with age (r = 0.31; P = 0.055 in AAF and r = 0.37; P = 0.066 in CF). Both lean and fat mass were major determinants of femoral neck BMD in AAF. Femoral BMD was directly related to body weight and body mass index in both races. Serum 25OHD and dehydroepiandrosterone sulfate approached statistical significance as independent predictors of femoral BMD in AAF. We conclude that in AAF, 1) bone density is higher than in CF, but there is a significant risk of fracture in a substantial number of subjects on the basis of BMD; 2) there is no difference in rates of age-related bone loss compared to those in CF; 3) both lean and fat mass are significant determinants of bone density; 4) serum estradiol and testosterone were higher than those in CF; and 5) aging is associated with increased bone turnover, 25OHD deficiency, and secondary hyperparathyroidism in both races. The absence of a difference in rates of bone loss between AAF and CF despite higher serum levels of PTH is compatible with the concept of a relative skeletal resistance to PTH in AAF.

Sequential activation of three distinct ICE-like activities in Fas-ligated Jurkat cells.

ICE family proteases have been implicated as important effectors of the apoptotic pathway, perhaps acting hierarchically in a protease cascade. Using cleavage of endogenous protease substrates as probes, three distinct tiers of ICE-like activity were observed after Fas ligation in Jurkat cells. The earliest cleavage detected (30 min) was of fodrin, and produced a 150 kDa fragment. The second phase of cleavage (50 min) involved PARP, U1-70kDa and DNA-PKcs, all substrates of the CPP32-like proteases. Lamin B cleavage was observed during the third cleavage phase (90 min). Distinct inhibition profiles obtained using a panel of peptide-based inhibitors of ICE-like proteases clearly distinguished the three different cleavage phases. These studies provide evidence for a sequence of ICE-like proteolytic activity during apoptosis. The early fodrin cleavage, producing a 150 kDa fragment, identifies an ICE-like activity proximal to CPP32 in Fas-induced Jurkat cell apoptosis.