Efficacy of genistein aglycone on some cardiovascular risk factors and homocysteine levels: A follow-up study.

BACKGROUND AND AIM: Recent evidence suggests that genistein aglycone may act beneficially on surrogate cardiovascular risk markers in postmenopausal women. We assessed the effects of genistein aglycone on some cardiovascular risk factors and homocysteine levels after 3-years of continued therapy in a cohort of osteopenic, postmenopausal women. METHODS AND RESULTS: The parent study was a randomized, double-blind, placebo-controlled trial involving 389 postmenopausal women with low bone mass for 24 months. Subsequently, a subcohort (138 patients) continued therapy for an additional year. Participants received 54mg of genistein aglycone (n=71) or placebo (n=67), daily. Both arms received calcium and vitamin D(3) in therapeutic doses. Moreover, 4 weeks before randomization procedures and during our follow-up study, all patients received dietary instructions in an isocaloric fat-restricted diet. Blood lipid profiles, fasting glucose and insulin, insulin resistance (HOMA-IR), fibrinogen, osteoprotegerin (OPG) and homocysteine at baseline and after 24 and 36 months of treatment were measured. Compared to placebo, genistein significantly decreased fasting glucose and insulin, HOMA-IR, fibrinogen and homocysteine after 24 and 36 months of treatment. By contrast, isoflavone administration did not affect high-density lipoprotein cholesterol and triglycerides though serum OPG was higher in the genistein recipients. There were no differences in adverse events or discomfort between groups. Results on routine biochemical, liver function, and hematologic testing did not change over time in placebo or genistein group. CONCLUSIONS: After 3-years of treatment, genistein aglycone plus calcium, vitamin D(3) and a healthy diet showed positive effects on some cardiovascular risk factors and homocysteine levels in a cohort of postmenopausal women with low bone mass.

A medicinal extract of Scutellaria baicalensis and Acacia catechu acts as a dual inhibitor of cyclooxygenase and 5-lipoxygenase to reduce inflammation.

A mixed extract containing two naturally occurring flavonoids, baicalin from Scutellaria baicalensis and catechin from Acacia catechu, was tested for cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) inhibition via enzyme, cellular, and in vivo models. The 50% inhibitory concentration for inhibition of both ovine COX-1 and COX-2 peroxidase enzyme activities was 15 microg/mL, while the mixed extract showed a value for potato 5-LOX enzyme activity of 25 microg/mL. Prostaglandin E2 generation was inhibited by the mixed extract in human osteosarcoma cells expressing COX-2, while leukotriene production was inhibited in both human cell lines, immortalized THP-1 monocyte and HT-29 colorectal adenocarcinoma. In an arachidonic acid-induced mouse ear swelling model, the extract decreased edema in a dose-dependent manner. When arachidonic acid was injected directly into the intra-articular space of mouse ankle joints, the mixed extract abated the swelling and restored function in a rotary drum walking model. These results suggest that this natural, flavonoid mixture acts via "dual inhibition" of COX and LOX enzymes to reduce production of pro-inflammatory eicosanoids and attenuate edema in an in vivo model of inflammation.

Ca2+ dependence of tension and ADP production in segments of chemically skinned muscle fibers.

Both ADP production and tension have been measured in segments of chemically skinned fibers contracting at different Ca2+ concentrations. Full mechanical activation occurred between pCa 7.00 and pCa 6.50. The total ATPase was due to both actomyosin and non-actomyosin ATPase. Actomyosin ATPase was observed at pCa 7.09 without accompanying tension. The Ca2+ dependence of tension was steeper than actomyosin ATPase. This finding implies some rate constants of the mechano-chemical cycle are Ca2+ dependent. Non-actomyosin ATPase was measured in fibers stretched beyond overlap of the thick and thin filaments. Sarcoplasmic reticulum was isolated and sarcoplasmic reticulum activity was measured in vitro under the same conditions as the single-fiber experiments. Non-actomyosin ATPase in the single fibers was found to be small compared to maximally activated actomyosin ATPase but larger than the ATPase that could be attributed to sarcoplasmic reticulum activity.

Effect of anisotropy and anharmonicity on protein crystallographic refinement. An evaluation by molecular dynamics.

Molecular dynamics simulations are employed to determine the errors introduced by anharmonicity and anisotropy in the structure and temperature factors obtained for proteins by refinement of X-ray diffraction data. Simulations (25 ps and 300 ps) of metmyoglobin are used to generate time-averaged diffraction data at 1.5 A resolution. The crystallographic restrained-parameter least-squares refinement program PROLSQ is used to refine models against these simulated data. The resulting atomic positions and isotropic temperature factors are compared with the average structure and fluctuations calculated directly from the simulations. It is found that significant errors in the atomic positions and fluctuations are introduced by the refinement, and that the errors increase with the magnitude of the atomic fluctuations. Of particular interest is the fact that the refinement generally underestimates the atomic motions. Moreover, while the actual fluctuations go up to a mean-square value of about 5 A2, the X-ray results never go above approximately 2 A2. This systematic deviation in the motional parameters appears to be due to the use of a single-site isotropic model for the atomic fluctuations. Many atoms have multiple peaks in their probability distribution functions. For some atoms, the multiple peaks are seen in difference electron density maps and it is possible to include these in the refinement as disordered residues. However, for most atoms the refinement fits only one peak and neglects the rest, leading to the observed errors in position and temperature factor. The use of strict stereochemical restraints is inconsistent with the average dynamical structure; nevertheless, refinement with tight restraints results in structures that are comparable to those obtained with loose restraints and better than those obtained with no restraints. The results support the use of tight stereochemical restraints, but indicate that restraints on the variation of temperature factors are too restrictive.

Determining local conformational variations in DNA. Nuclear magnetic resonance structures of the DNA duplexes d(CGCCTAATCG) and d(CGTCACGCGC) generated using back-calculation of the nuclear Overhauser effect spectra, a distance geometry algorithm and constrained molecular dynamics.

Two-dimensional nuclear magnetic resonance (n.m.r.) spectroscopy and a variety of computational techniques have been used to generate three-dimensional structures of the two DNA duplexes d(CGCCTAATCG) and d(CGTCACGCGC). The central six base-pairs in these two decamers contain all ten dinucleotide pairs in DNA and thus, represent a model system for investigating how the local structure of DNA varies with base sequence. Resonance assignments were made for the non-exchangeable base protons and most of the C-1'-C-4' sugar protons in both decamers. Three-dimensional structures were generated using a distance geometry algorithm and these initial structures were refined by optimizing the fit of back-calculated spectra against the experimental two-dimensional nuclear Overhauser effect (NOE) spectra. This back-calculation procedure consists of calculating NOE cross relaxation rates for a given structure by solution of the Bloch equations, and directly accounts for spin diffusion effects. Use of this refinement procedure eliminates some assumptions that have been invoked when generating structures of DNA oligomers from n.m.r. data. Constrained energy minimization and constrained quenched molecular dynamics calculation were also performed on both decamers to help generate energetically favorable structures consistent with the experimental data. Analysis of the local conformational parameters of helical twist, helical rise, propeller twist, displacement and the alpha, beta, gamma, epison and zeta backbone torsion angles in these structures shows that these parameters span a large range of values relative to the X-ray data of nucleic acids. However, the glycosidic and pseudorotation angles are quite well defined in these structures. The implications that these results have for determination of local structural variations of DNA in solution, such as those predicted by Callidine's rules, are discussed. Our results differ significantly from some previous studies on determining local conformations of nucleic acids and comparisons with these studies are made.

Temperature-dependent molecular dynamics and restrained X-ray refinement simulations of a Z-DNA hexamer.

Molecular dynamics simulations of the Z-DNA hexamer 5BrdC-dG-5BrdC-dG-5BrdC-dG were performed at several temperatures between 100 K and 300 K. Above 250 K, a strong sequence-dependent flexibility in the nucleic acid is observed, with the guanine sugar and the phosphate of GpC sequences much more mobile than the cytosine sugar and phosphate of CpG sequences. At 300 K, the hexamer is in dynamic equilibrium between several Z forms, including the crystallographically determined ZI and ZII forms. The local base-pair geometry, however, is not very variable, except for the roll of the base-pairs. The hexamer molecular dynamics trajectories have been used to test the restrained parameter crystallographic refinement model for nucleic acids. X-ray diffraction intensities corresponding to observed diffraction data were computed. The average structures obtained from the simulations were then refined against the calculated intensities, using a restrained least-squares program developed for nucleic acids in order to analyse the effects of the refinement model on the derived quantities. In general, the temperature dependence of the atomic fluctuations determined directly from the refined Debye-Waller factors is in reasonably good agreement with the results obtained by calculating the atomic fluctuations directly from the Z-DNA molecular dynamics trajectories. The agreement is best for refinement of temperature factors without restraints. At the highest temperature studied (300 K), the effect of the refinement on the most mobile atoms (phosphates) is to significantly reduce the mean-square atomic fluctuations estimated from the refined Debye-Waller factors below the actual values (less than (delta r)2 greater than congruent to 0.5 A2). Analysis of the temperature-dependence of the mean-square atomic fluctuations provides information concerning the conformational potential within which the atoms move. The calculated temperature-dependence and anharmonicity of the Z-DNA helix are compared with the results observed for proteins. The average structures from the simulations were refined against the experimental X-ray intensities. It is found that low-temperature molecular dynamics simulations provide a useful tool for optimizing the refinement of X-ray structures.

Colonisation rates of Streptococcus pyogenes and Staphylococcus aureus in the oropharynx of a young adult population.

There are very few reports on the rates of oropharyngeal colonisation by Streptococcus pyogenes and Staphylococcus aureus in young adults. The present study found colonisation rates of 9.6% and 26.2%, respectively. These rates are two-fold higher than historical rates, indicating that these organisms may be more prevalent than thought previously. This finding may have important clinical consequences in certain populations, and requires further investigation.

Performance feedback deficit in geriatric depression.

BACKGROUND: The orbital frontal cortex is involved with processing of performance feedback. This study tests the hypothesis that older depressed subjects, compared with elderly control subjects, commit more subsequent errors after receiving feedback from an initial error. METHODS: We administered 116 older depressed patients and 139 control subjects the Trail Making Test Part B (TRAILS-B). Subjects who committed an error on TRAILS-B were immediately given feedback on performance. We then measured the frequency of making an error on the subsequent three tries. The likelihood of making any subsequent error was examined. RESULTS: After controlling for the overall initial error rate, more depressed patients than control subjects made subsequent errors. This association remained significant in later regression models. When the depressed group was examined in additional models, severity of depression was not associated with increased subsequent errors. CONCLUSIONS: These results extend previous findings suggesting a performance feedback deficit in geriatric depression. The findings support previous studies linking the orbital frontal cortex and depression.

Central nervous system dysfunction in acquired immunodeficiency syndrome.

Nearly 40% of AIDS patients develop neurological complications during the course of their illness, and about 10% experience neurological symptoms as the initial manifestations of AIDS. The most common neurological complication (14% of AIDS patients) is human immunodeficiency virus (HIV) encephalopathy, but opportunistic viral and nonviral infections and neoplasms are also quite common; the most frequent among these are cryptococcal meningitis, toxoplasmosis, primary central nervous system (CNS) lymphoma, progressive multifocal leukoencephalopathy, and herpesvirus infections. Most of the nonviral infections and neoplasms are potentially treatable. Neurological syndromes include diffuse and regional encephalopathies, myelopathy, meningitis, intraaxial cranial neuropathies, and retinopathy. About 10% of AIDS patients develop a CNS mass lesion; the chief causes of these lesions are toxoplasmosis and primary CNS lymphoma. Since the clinical profiles of the various diseases overlap to a great extent, differential diagnosis requires a thorough workup, including magnetic resonance imaging or computed tomography brain scanning, examination of the cerebrospinal fluid, and, frequently, brain biopsy. Because AIDS patients have a high incidence of multiple intracranial pathologies, the diagnostic workup may have to be repeated to identify all of the diseases present.

Progressive multifocal leukoencephalopathy in patients with HIV infection: lack of impact of early diagnosis by stereotactic brain biopsy.

Thirteen patients with HIV-related progressive multifocal leukoencephalopathy (PML), representing an institutional incidence of 4.2%, are reported. All cases were diagnosed by image guided stereotactic brain biopsy shortly after their presentation for neurologic complaints. All patients were males; risk factors included homosexual or bisexual activity or intravenous drug use. At the time of presentation with PML, the mean T4 count was 85 (range 9-240 cells/mm3). The most common neurologic symptoms were cognitive dysfunction and aphasia, whereas gait abnormalities and disordered cognition were the most common neurologic signs. Cerebrospinal fluid analysis was helpful only to rule out other causes of CNS disease. Magnetic resonance imaging, more sensitive than computed tomography (CT) scanning, typically revealed multiple areas of increased intensity on T2 weighted images although unifocal disease was seen in 23% of patients. Despite early stereotactic biopsy and aggressive symptomatic therapy, survival of these patients was poor with a mean of 2.6 months after the onset of neurological symptoms and 2.0 months after biopsy.

MRI-guided stereotaxic brain biopsy in neurologically symptomatic AIDS patients.

Two patients with AIDS-related neurologic dysfunction were evaluated with both computed tomographic (CT) brain scans and magnetic resonance imaging (MRI). CT scans were essentially normal in both patients while MRI revealed focal lesions amenable to brain biopsy. Using newly developed instrumentation, MRI-guided stereotaxic brain biopsy was performed without complication. The benefits and impact of this new technology for the care of neurologically symptomatic AIDS patients is discussed.

Neuroepidemiology of acquired immunodeficiency syndrome.

Data from the Centers for Disease Control (CDC) and from the hospitals affiliated with the University of California, San Francisco show a significant incidence of neurological complications in AIDS patients and suggest that patients from different risk groups and geographic regions are at different relative risk for specific neurological complications. CDC national surveillance data show that Haitian-born AIDS patients are 3.7 times more likely to have neurological complications than are patients in other risk groups; neurological illness is also reported more often in intravenous drug abusers and black AIDS patients. Cryptococcal meningitis is most prevalent among intravenous drug abusers, Haitians, and blacks, and is most commonly reported in New Jersey, a state with a large proportion of AIDS patients in these three groups. Cerebral toxoplasmosis is reported much more often in Haitians than in other risk groups and is most prevalent in Florida among both Haitians and non-Haitians, probably because of greater exposure to Toxoplasma gondii organisms in the semitropical climate of Florida. The prevalence rates for progressive multifocal leukoencephalopathy (PML) and primary central nervous system lymphoma are similar throughout various risk groups and regions of the United States.

The efficacy and clinical impact of brain imaging in neurologically symptomatic AIDS patients: a prospective CT/MRI study.

The relative efficacies of cranial magnetic resonance imaging (MRI) and computed tomography (CT) brain scans for the detection of intracranial pathology in patients with the acquired immune deficiency syndrome (AIDS) were evaluated prospectively. Fifty homosexual or bisexual men with AIDS and neurologic symptoms were evaluated using both modalities. In 24 patients, MR images and CT scans provided the same diagnostic information (within normal limits in 16, cerebral atrophy only in 6, and similar lesions in 2 patients). In only one instance did CT show the presence of a lesion not seen on MRI. In the 25 remaining patients, MRI was the more sensitive modality. MRI also reflected more consistently the histopathologically documented extent and distribution of central nervous system disease. The greater sensitivity of MRI suggested significant alterations in the diagnostic evaluation and therapeutic management of 20 patients. Thus, MRI was as good or better than CT for the detection of intracranial pathology in 49 of 50 neurologically symptomatic AIDS patients and significantly affected the diagnosis and treatment of 40% of these patients. Although MRI does not appear to be more specific than other modalities in the differentiation of human immunodeficiency virus (HIV)-related neurologic diseases, its greater sensitivity suggests that MRI may be the best neuroimaging procedure for the initial radiologic evaluation of AIDS patients with neurologic illness.

Proton nuclear magnetic resonance imaging of acute experimental cerebral ischemia.

To determine the efficacy of proton nuclear magnetic resonance (NMR) imaging in documenting acute ischemic infarction and to characterize the changes in these images during the first 24 hours following the ischemic insult, serial NMR imaging was performed in gerbils that had undergone unilateral carotid artery ligation. No significant changes in the signal intensity, T1 or T2 relaxation times were noted for either asymptomatic animals or the control hemisphere of symptomatic gerbils. There was a significant increase in T1 and especially T2 relaxation times and in both the relative signal intensity and Hf(v) for the ischemic hemisphere of symptomatic gerbils. These parameters appeared to increase linearly over 24 hours. The ischemic lesion first could be detected by NMR as early as 3 hours after carotid artery ligation, our earliest time point. The physiologic significance of these changes is discussed. These data suggest that NMR imaging may have significant diagnostic importance for acute cerebral ischemia and infarction in man.

Computer simulations of macromolecular dynamics: models for vibrational spectroscopy and X-ray refinement.

The use of computer simulations to study the internal dynamics of globular proteins and nucleic acids has attracted a great deal of attention in recent years. These simulations constitute the most detailed theoretical approach available for studying internal motions and structural flexibility of biological macromolecules. In this paper we review recent work concerned with the use of computer simulations for the interpretation of, and comparison with, experimental probes of molecular dynamics. New methods for calculating vibrational spectroscopic lineshapes from computer simulations are discussed. A quasiharmonic approximation is described by which classical computer simulations on multidimensional potential surfaces can be used to estimate the effects of anharmonicity on vibrational spectra. A novel aspect of the method is the use of normal-mode eigenvectors as the independent coordinates for Monte Carlo sampling. Results for isolated small molecules and liquid water are reviewed. The construction of vibrational lineshapes from quantum computer simulations using path integral methods with new (quasiharmonic and variable quadratic) reference systems are discussed. Results for small model systems are presented and extension of the methods to large molecules is discussed. The recent use of molecular dynamics simulations to analyze X-ray refinement models for proteins and nucleic acids is also reviewed.

The effect of analgesic doses of morphine on regional cerebral glucose metabolism in pain-related structures.

The effect of morphine on regional cerebral glucose metabolism was measured in rats using high resolution [14C]2-deoxyglucose autoradiography with concurrent confirmation of morphine-induced analgesia measured by tail-flick latency to noxious heat. Within the limits of resolution of this technique, doses of morphine sufficient to inhibit the tail-flick reflex had no significant effect on glucose metabolism in structures implicated in the modulation of pain.

Comparison of cytosine arabinoside delivery to rat brain by intravenous, intrathecal, intraventricular and intraparenchymal routes of administration.

We evaluated the delivery of 14C-cytosine arabinoside (AraC) to rat brain by: 1) intravenous (IV) bolus, by 2) intrathecal (IT) and 3) intraventricular (IVT) infusion, and by 4) convection-enhanced delivery (CED) into the caudate nucleus. Plasma and brain AraC metabolites were measured with HPLC, and distribution and concentration of 14C-AraC in brain sections were measured by quantitative autoradiography. After IV administration, the alpha and beta plasma half-lives were 1.9 and 46.5 min, respectively. The blood-to-brain transfer constant of AraC was 2.5+/-1.4 microliter g(-1) min(-1), compatible with high water solubility. After IT and IVT administration, tissue levels were high at the brain and ventricular surfaces, but declined exponentially into brain. After CED, maximum brain levels were up to 10,000 times higher than the IV group, and the distribution pattern was one of high 14C-AraC concentration in the convective component, with exponentially declining concentrations outside this region. The rate loss constant from brain was 0.002+/-0.0004 min(-1), suggesting that AraC was accumulating in brain cells. AraC was metabolized into uracil arabinoside within the brain. 14C-AraC was infused into 1 dog and distributed widely in the ipsilateral hemisphere. These studies suggest that delivery of AraC to brain parenchyma by the IV, IT or IVT routes will be subtherapeutic. Delivery by CED can achieve, and maintain, therapeutic levels of AraC in the brain, and should be further evaluated as a potential method of drug delivery.

Changes in blood-brain barrier permeability associated with insertion of brain cannulas and microdialysis probes.

Blood-brain barrier (BBB) transcapillary transport was studied after insertion of cannulas and microdialysis probes into the brains of three groups of rats. Quantitative autoradiography was used to measure changes in BBB permeability around the insertion site. In the first group, BBB function was measured with 14C-sucrose at times from immediately, and up to 28 days, after insertion of a microdialysis probe. BBB function was disrupted biphasically: a 19-fold increase in the influx constant (K1) of sucrose occurred immediately after insertion with a second 17-fold increase at 2 days, followed by a slow decline to 5 times normal values at 28 days. In the second group, 14C-dextran (70 kDa) was used to measure BBB transcapillary transport; K1 was increased 90-fold after probe insertion. In the 3rd group, 14C-AIB (alpha-aminoisobutyric acid) was used to evaluate BBB transport after insertion of a 27 gauge cannula, which was used to infuse 1 microliter of saline over 5 min. The K1 of AIB was increased 25 times control values. We conclude that BBB transcapillary transport function is disturbed in response to insertion of brain cannulas and/or microdialysis probes, that BBB dysfunction is maximal at the cannula or probe tip, varies with time after insertion, may persist for at least 28 days after insertion, and occurs over a wide molecular range of solutes. These results suggest caution when using microdialysis as a method to study normal BBB function, and suggest that microdialysis may overestimate the rate of transfer into and out of the brain.