Pulsed light and antimicrobial combination treatments for surface decontamination of cheese: Favorable and antagonistic effects.

Postprocessing cross-contamination of cheese can lead to both food safety issues and significant losses due to spoilage. Pulsed light (PL) treatment, consisting of short, high-energy, broad-spectrum light pulses, has been proven effective in reducing the microbial load on cheese surface. As PL treatment effectiveness is limited by light-cheese interactions, the possibility to improve its effectiveness by combining it with the antimicrobial nisin was explored. The effect of natamycin, which is added to cheeses as an antifungal agent, on PL effectiveness was also investigated. Pseudomonas fluorescens, Escherichia coli ATCC 25922, and Listeria innocua were used as challenge microorganisms. Bacterial cultures in stationary growth phase were diluted to initial inoculum levels of 5 or 7 log cfu per cheese slice. Slices of sharp white Cheddar cheese and white American singles were cut in rectangles of 2.5 × 5 cm. For cheese slices receiving antimicrobial treatment before PL, slices were dipped in natamycin or nisin, spot inoculated with 100 µL of bacterial suspension, and then treated with PL. Cheese slices receiving PL treatment before antimicrobials were spot inoculated, treated with PL, and then treated with antimicrobials. The PL fluence levels from 1.02 to 12.29 J/cm2 were used. Survivors were enumerated by standard plate counting or the most probable number technique, as appropriate. All treatments were performed in triplicate, and the data were analyzed using a general linear model. Treatment with nisin or natamycin before PL decreased the effectiveness of PL for all bacteria tested. For instance, PL reduced P. fluorescens on Cheddar cheese by 2.19 ± 0.27 log after 6.14 J/cm2, whereas combination treatments at the same PL fluence yielded barely 1 log reduction. Inactivation of L. innocua on Cheddar was only 0.78 ± 0.01 log when using PL after nisin, compared with a 1.30 ± 0.76 log reduction by nisin alone. This was attributed to the absorption of UV light by the 2 antimicrobials, which diminished the UV fluence received by the bacteria. Increased inactivation was obtained when antimicrobials were applied after PL. On process cheese, a maximum reduction of 3.73 ± 0.96 log of L. innocua was obtained at 9.22 J/cm2 for PL followed by nisin, compared with 3.01 ± 0.48 by PL alone. This study demonstrates that antimicrobials may increase the antimicrobial effectiveness of PL on cheese surface, but the order of treatments is critical.

Pulsed-light inactivation of pathogenic and spoilage bacteria on cheese surface.

Cheese products are susceptible to postprocessing cross-contamination by bacterial surface contamination during slicing, handling, or packaging, which can lead to food safety issues and significant losses due to spoilage. This study examined the effectiveness of pulsed-light (PL) treatment on the inactivation of the spoilage microorganism Pseudomonas fluorescens, the nonenterohemorrhagic Escherichia coli ATCC 25922 (nonpathogenic surrogate of Escherichia coli O157:H7), and Listeria innocua (nonpathogenic surrogate of Listeria monocytogenes) on cheese surface. The effects of inoculum level and cheese surface topography and the presence of clear polyethylene packaging were evaluated in a full factorial experimental design. The challenge microorganisms were grown to early stationary phase and subsequently diluted to reach initial inoculum levels of either 5 or 7 log cfu/slice. White Cheddar and process cheeses were cut into 2.5×5 cm slices, which were spot-inoculated with 100 µL of bacterial suspension. Inoculated cheese samples were exposed to PL doses of 1.02 to 12.29 J/cm(2). Recovered survivors were enumerated by standard plate counting or the most probable number technique, as appropriate. The PL treatments were performed in triplicate and data were analyzed using a general linear model. Listeria innocua was the least sensitive to PL treatment, with a maximum inactivation level of 3.37±0.2 log, followed by P. fluorescens, with a maximum inactivation of 3.74±0.8 log. Escherichia coli was the most sensitive to PL, with a maximum reduction of 5.41±0.1 log. All PL inactivation curves were nonlinear, and inactivation reached a plateau after 3 pulses (3.07 J/cm(2)). The PL treatments through UV-transparent packaging and without packaging consistently resulted in similar inactivation levels. This study demonstrates that PL has strong potential for decontamination of the cheese surface.

Cerebral hemodynamics and baroreflex sensitivity after carotid artery stenting.

OBJECTIVES: The long-term hemodynamic effects of carotid angioplasty and stenting (CAS) are unclear. We performed a longitudinal study to investigate the variations in cerebral hemodynamics in patients undergoing CAS. MATERIALS AND METHODS: We performed prospective evaluation of 63 symptomatic male patients (19 patients had transient ischemic attack and 44 had minor stroke; mean age: 77.3 ± 6.3 years [range: 51-86]). The mean blood flow velocities (MBFV) and pulsatility index (PI) of the middle cerebral arteries (MCA) on both sides were evaluated using transcranial color-coded Doppler (TCCD) ultrasonography. Cardiac autonomic activities were evaluated by measuring baroreflex sensitivity (BRS). All parameters were measured at baseline prior to CAS and at 1, 3, 6, and 12 months after CAS. RESULTS: The preoperative MBFV and PI of the ipsilateral MCA were significantly lower than those of the contralateral side. However, after CAS, MBFV in the ipsilateral MCA increased significantly until 2 weeks after stenting, after which the MBFV gradually decreased and remained stable for 1 year after CAS. Further, we observed a nonsignificant increase in MBFV in the contralateral MCA after CAS. In contrast to the MBFV, the BRS values decreased significantly 1 month after stenting and returned to baseline levels 6 months after CAS. CONCLUSIONS: Patients with CAS showed improved global cerebral hemodynamic status. However, the BRS did not normalize initially, and baseline value was achieved at 6 months after stenting.

Contribution of intracranial vertebral artery asymmetry to vestibular neuropathy.

OBJECTIVES: To test the hypothesis that vertebral artery hypoplasia (VAH) may affect the lateralisation of vestibular neuropathy (VN), probably through haemodynamic effect on the vestibular labyrinth. METHODS: 69 patients with unilateral VN were examined with a magnetic resonance angiographic (MRA) and caloric test. 50 healthy subjects served as controls. The diagnosis of intracranial VAH was based on MRA if <0.22 cm in VA diameter and a diameter asymmetry index >40%. The authors then correlated the canal paretic side with the VAH side. RESULTS: MRA study revealed 29 VAH (right/left: 23/6) in VN subjects and six VAH in controls (right/left: 5/1). The RR of VAH in VN subjects compared with controls was elevated (RR=2.2; 95% CI 1.8 to 2.8). There was a high accordance rate between the side of VAH and VN. Among 29 patients with unilateral VAH, 65.5% (N=19) had an ipsilateral VN, in which left VAH showed a higher accordance rate (83.3%) than the right side (60.9%). VN subjects with vascular risk factors also had a higher VAH accordance rate (81%) than those without (25%). CONCLUSIONS: VAH may serve as a regional haemodynamic negative contributor and impede blood supply to the ipsilateral vestibular labyrinth, contributing to the development of VN, which could be enhanced by atherosclerotic risk factors and the left-sided location.

Cr(VI) removal on fungal biomass of Neurospora crassa: the importance of dissolved organic carbons derived from the biomass to Cr(VI) reduction.

Interactions of toxic Cr(VI) with renewable biomaterials are considered an important pathway for Cr(VI) removal in ecosystems. Biomaterials are susceptible to dissolution, and their dissolved derivatives may provide an alternative to surface-involved pathway for scavenging of Cr(VI). In this study, dissolved organic carbon (DOC) derived from Neurospora crassa biomass was investigated. The proportion of Cr(VI) reduction by DOC to that on biomass was determined to evaluate the importance of DOC to Cr(VI) reduction. A rapid increase in DOC concentration from 145.6 to 193.7 mg L(-1) was observed when N. crassa-biomass was immersed in 0.01 M KCl solution at pH of 1-5, and polysaccharides, peptides, and glycoproteins with carboxyl, amide, and -NH functional groups, are the major compositions of DOC. On reaction of 96.2 microM Cr(VI) with N. crassa-biomass or DOC, it was estimated that DOC contributed approximately 53.8-59.5% of the total Cr(VI) reduction on biomass in the dark. Illumination enhanced Cr(VI) reduction via photo-oxidation of biomass/DOC under aeration conditions, which formed superoxide for Cr(VI) reduction. At pH 1, photoinduced Cr(VI) reduction by DOC proceeded more rapidly than reduction on the biomass surface. However, at pH >3, with a decrease in Cr(VI) reduction by DOC, photon-excited biomass may become an important electron source for Cr(VI) photoreduction.

N-Methyl-D-aspartate receptor antagonist MK-801 attenuates morphine tolerance and associated glial fibrillary acid protein up-regulation: a proteomic approach.

BACKGROUND: It is well known that long-term morphine administration results in tolerance, which limits the clinical use of this drug in pain management. METHODS: Male Wistar rats were randomly assigned to receive one of four different infusions: morphine [15 microg/h, intrathecal (i.t.)], saline, MK-801 (5 microg/h, i.t.) plus morphine (15 microg/h, i.t.), or MK-801 (5 microg/h, i.t.) alone. RESULTS: Morphine infusion induced a maximal antinociceptive effect on day 1 and tolerance on day 3, and the maximal anti-receptive tolerance was observed on day 5. Co-infusing MK-801 with morphine attenuated morphine's anti-receptive tolerance. Two-dimensional gel electrophoretic analysis of spinal proteins revealed that eight protein spots were up-regulated in morphine-tolerant rats, and that they were significantly inhibited by MK-801 co-infusion. Among the up-regulated proteins, glial fibrillary acid protein (GFAP), a glial-specific maker, was identified by mass spectrometry. This finding was also confirmed by Western blot analysis. CONCLUSION: Using proteomic analysis, we identified eight GFAP protein spots that were up-regulated in the dorsal horn of morphine-tolerant rat spinal cords. This up-regulation was partly inhibited by N-methyl-D-aspartate receptor antagonist MK-801 co-infusion, which suggests that GFAP protein can be considered to be a pathogenesis marker of morphine tolerance.

Gene amplification and overexpression of protein phosphatase 1alpha in oral squamous cell carcinoma cell lines.

Gene amplification of chromosomal band 11q13 is observed frequently in oral squamous cell carcinomas (OSCC). Several genes have been identified in the 11q13 amplicon, including FGF3, FGF4, CCND1, EMS1 and TAOS1. Some of these genes show good correlation between gene copy number and gene expression, and are thought to play a role in driving 11q13 amplification. The PPP1CA gene, which encodes the catalytic subunit of serine/threonine protein phosphatase protein phosphatase 1alpha (PP1alpha), is also located in 11q13. Protein phosphatase 1alpha, one of the isoforms of PP1, regulates critical cellular events, such as cell cycle progression, and apoptosis. We sought to explore the possibility that PPP1CA was amplified and overexpressed in OSCC cells. Indeed, some OSCC cell lines had PPP1CA gene amplification, as analysed by fluorescence in situ hybridization. We have also demonstrated that PPP1CA gene copy number is increased in 21% of the OSCC cell lines determined by quantitative microsatellite analysis. PP1alpha RNA expression determined by quantitative reverse transcription-polymerase chain reaction was significantly higher in OSCC cell lines with 11q13 amplification compared to those without 11q13 amplification (P=0.011). The difference was even more significant between cell lines with at least three copies of the PPP1CA gene and those with less than three copies of the gene (P=0.00045). Relative PP1alpha protein levels were also significantly associated with PPP1CA gene copy number (P=0.014). Furthermore, knockdown of PP1alpha and/or cyclin D1 by small interfering RNA suppressed OSCC cell growth, at least in part by modulating pRB phosphorylation, resulting in G0 growth arrest. These data suggest that like the cyclin D1 gene, CCND1, amplification and overexpression of the PP1alpha gene, PPP1CA, may be involved in OSCC tumorigenesis and/or progression.

Characterization of the extracellular bactericidal factors of rat alveolar lining material.

The surfactant fraction (55,000-g pellet) of leukocyte-free rat bronchoalveolar lavage fluid contains factors that rapidly kill and lyse pneumococci. These factors were purified and identified biochemically by using a quantitative bactericidal test to monitor fractionation procedures. 91% of the antipneumococcal activity of rat surfactant was recovered in chloroform after extraction of rat surfactant with chloroform-methanol (Bligh-Dyer procedure). After chromatography on silicic acid with chloroform, acetone, and methanol, all detectable antibacterial activity (approximately 80% of the initial activity) eluted with the neutral lipids in chloroform. When rechromatographed on silicic acid with hexane, hexane-chloroform, and chloroform, the antibacterial activity eluted with FFA. Thin-layer chromatography (TLC) established that the antibacterial activity was confined to the FFA fraction. Gas-liquid chromatography showed that the fatty acid fraction contained a mixture of long-chain FFA (C12 to C22) of which 66.7% were saturated and 32.4% were unsaturated. The quantity of TLC-purified FFA needed to kill 50% of 10(8) pneumococci under standardized conditions (one bactericidal unit) was 10.6 +/- 0.5 micrograms. Purified FFA acted as detergents, causing release of [3H]choline from pneumococcal cell walls and increased bacterial cell membrane permeability, evidenced by rapid unloading of 3-O-[3H]methyl-D-glucose. FFA acting as detergents appear to account for the bactericidal and bacteriolytic activity of rat pulmonary surfactant for pneumococci.

The removal and recovery of Cr(VI) by Li/Al layered double hydroxide (LDH).

Hexavalent Cr has been identified as one of the toxic metals commonly present in industrial effluents. Among the treatment techniques developed for removing Cr(VI) from waste waters, sorption is most commonly applied, due to its simplicity and efficiency. However, few adsorbents can be recycled and reused cost-effectively. In this study, the removal and recovery of Cr(VI) from water using Li/Al LDH was investigated. The removal of Cr(VI) by Li/Al LDH was evaluated in a batch mode. The results demonstrated that Cr(VI) adsorption onto Li/Al LDH occurs by replacing the Cl(-) that originally exists in the interlayer of the adsorbent. The degree of Cr(VI) adsorption observed for Li/Al LDH was relatively high and the process occurred rapidly; however, a portion of adsorbed Cr(VI) was gradually desorbed, due to the Li de-intercalation of Li/Al LDH. Lithium de-intercalation from Li/Al LDH with interlayer Cl(-) and interlayer Cr(VI) follows the first order kinetics and has the activation energies of 76.6 and 41.5 kJ mol(-1), respectively. The properties of thermal unstability and the high adsorption capacity of Li/Al LDH may lead to the development of an innovative technique for the removal of Cr(VI) from Cr(VI)-containing wastewater. That is, Li/Al LDH may be used as an effective adsorbent for the adsorption of Cr(VI) in an ambient environment. Following the adsorptive process, the adsorbed Cr(VI) may be released, using heated water to treat the Cr(VI)-containing Li/Al LDH particles. Through this hydrothermal treatment of the used adsorbent, Cr(VI) can be recovered and the solid product (gibbsite) can be recycled for further use.

Cloning and characterization of overexpressed genes in the mammary gland of rat strains carrying the mammary carcinoma suppressor (Mcs) gene.

Inbred rat strains vary in their susceptibilities to mammary carcinogenesis. The Copenhagen (COP) and Wistar-Kyoto (WKY) rats are tumor resistant, whereas the Wistar-Furth (WF), Fischer (F344), and outbred Sprague-Dawley (SD) rats are susceptible. A dominant pattern of inheritance acting via the mammary carcinoma suppressor (Mcs) gene(s), which is mainly responsible for mammary tumor resistance, has been defined in the COP and WKY rats. In order to understand the basis of the phenotype, COP and WF mammary mRNAs were used for subtractive hybridization to isolate genes associated with the activity of the Mcs gene(s). Three genes, alpha-casein, lipoprotein lipase, and an unidentified gene, were found to be overexpressed in the mammary gland of the COP rat. In addition to alpha-casein overexpression, Northern analysis demonstrated that beta- and gamma-casein genes were also highly expressed in the mammary glands of tumor-resistant WKY and COP virgin rats but not the susceptible F344, WF, and SD strains. The association of casein gene expression with the tumor-resistant phenotype was further investigated by determining the functional site of the strain-specific casein gene regulation by using a mammary cell transplantation assay. In contrast to its normal endocrine control during pregnancy and lactation, casein gene overexpression was found to be controlled within the mammary epithelial cells of virgin rats. This is also the site of production and action of the Mcs gene product. Comparison of polymerase chain reaction-amplified beta-casein precursor RNA levels with the use of reverse transcription-polymerase chain reaction revealed that the regulation of this gene is likely at the transcriptional level. These data suggest an association of overexpression of casein genes, with the Mcs phenotype. The biological significance of this association is under investigation.

Reduction of dimesna to mesna by the isolated perfused rat liver.

Mesna is administered with ifosfamide and cyclophosphamide to reduce the incidence of hemorrhagic cystitis. In the present model of mesna metabolism and disposition, mesna is rapidly and irreversibly oxidized to dimesna in the plasma, passes unchanged through the liver, and is then reduced by the kidney and excreted. Our detection of a high ratio of mesna to dimesna in the plasma of clinical samples led us to reinvestigate the hepatic metabolism of mesna and dimesna. We perfused isolated rat livers from female Sprague Dawley rats with protein-free buffered solution containing dimesna at concentrations observed during therapy. In single-pass perfusions, each liver was perfused with up to three dimesna concentrations during consecutive 20-min periods. Recirculating perfusions were used to study single supratherapeutic concentrations of dimesna or mesna. Mesna and dimesna concentrations were measured by specific chromatographic procedures. Dimesna reduction, adjusted by the effluent flow rate and liver weight (0.4-58.5 nmol/min/g liver), correlated closely by linear regression (r = 0.98; n = 36) to the perfused dimesna concentration (4.2-249 microM), indicating a clearance of 0.20 ml/min/g liver. The concentration of dimesna that entered the liver closely matched the summed concentration of mesna and dimesna emerging in the effluent perfusate (single-pass experiments: slope, 0.98; intercept, -0.30; r = 1.00; n = 31). Only trace amounts of unidentified thiols were detected in the bile during recirculation of perfusates with 1 mM mesna or 250 microM dimesna. The effluent mesna concentration correlated inversely with the flow rate, which was consistent with a low extraction ratio in the perfusion model. These data suggested that the dimesna reduction rate was limited by hepatic uptake. Dimesna reduction was decreased by agents that deplete glutathione. Pretreatment of rats with up to 100 mg/kg ifosfamide did not impair hepatic dimesna reduction. In control experiments, dimesna was not reduced during recirculation through the apparatus without a liver. Mesna was oxidized to dimesna during oxygenation of the perfusate in the reservoir, but mesna injected directly into the perfusate just before entry into the liver passed unchanged into the effluent. Extrapolation of the dimesna clearance data from the perfusion model to humans suggests that hepatic dimesna reduction may counterbalance the rapid oxidation of mesna in plasma. The proposed equilibrium is consistent with clinical observations and suggests a new model for mesna metabolism and disposition.

Identification of a gamma-tubulin-binding domain in BRCA1.

We previously reported (L-C. Hsu and R. L. White, Proc. Natl. Acad. Sci. USA, 95: 12983-12988, 1998) that hypophosphorylated BRCA1 is associated with mitotic centrosomes in vivo, perhaps through its interaction with gamma-tubulin. In vitro evidence presented here indicates that full-length BRCA1 protein generated by in vitro translation interacts with gamma-tubulin. A specific domain of BRCA1 protein, BRCA1 fragment no. 3 (BF3; amino acids 504-803), is both necessary and sufficient to bind gamma-tubulin. BF3 and gamma-tubulin coimmunoprecipitated when coexpressed in cells. In addition, expression of BF3 interfered with the interaction between BRCA1 and gamma-tubulin. Stable transformants of COS-7 cells that overexpressed BF3 showed a reduced growth rate partly because of increased apoptosis. Furthermore, overexpression of BF3 in COS-7 cells results in the accumulation of mitotic cells with multiple centrosomes and abnormal spindles. Okadaic acid, an inhibitor of protein phosphatases types 1 and 2A, induces hyperphosphorylation of BRCA1, a reduction of both BRCA1 and gamma-tubulin associated with mitotic centrosomes, and an accumulation of abnormal spindle formation. Thus, attenuating the interaction between BRCA1 and gamma-tubulin, and their association with mitotic centrosomes, may induce an increase of aneuploid cell population and contribute to tumorigenesis.

Genetic identification of Mcs-1, a rat mammary carcinoma suppressor gene.

Women have inherited differences in their susceptibility to breast cancer, but the genes underlying this variation are difficult to identify. We have approached the problem of identifying breast cancer susceptibility genes by using a rat model. Inbred rat strains display differential susceptibilities to mammary carcinogenesis; the Copenhagen (COP) rat is resistant, while the Wistar-Furth (WF) rat is susceptible to induction of mammary tumors by 7,12-dimethylbenz[a]anthracene. Genetic breeding studies have shown that tumor resistance in the COP rat is a dominant phenotype, termed the rat mammary carcinoma suppressor trait. As a step toward defining the basis of this resistance, we undertook genetic mapping of this phenotype in a (WF x COP)F1 x WF backcross by studying a large collection of microsatellite and minisatellite polymorphisms. A total of 114 genetic markers, covering approximately 75% of the rat genome, were genotyped in the backcross progeny. A marker on rat chromosome 2 was found to show linkage to the resistance phenotype. Genetic linkage was demonstrated both in a qualitative analysis (in which rats were defined as resistant if they developed 0 tumors and sensitive if they developed two or more tumors; LOD score, 4.0) and in a quantitative trait locus analysis (in which tumor number was used as the quantitative phenotype; LOD score, 3.8). We infer the existence of a gene, Mcs-1, on rat chromosome 2 that suppresses mammary carcinogenesis.

Downregulation of COMMD1 by miR-205 promotes a positive feedback loop for amplifying inflammatory- and stemness-associated properties of cancer cells.

Sustained activation of nuclear factor-κB (NF-κB) in cancer cells has been shown to promote inflammation, expansion of cancer stem cell (CSC) population, and tumor development. In contrast, recent studies reveal that CSCs exhibit increased inflammation due to constitutive NF-κB activation; however, the underlying molecular mechanism remains unclear. In the present study, the analysis of microarray data revealed upregulation of NF-κB-regulated pro-inflammatory genes and downregulation of copper metabolism MURR1 domain-containing 1 (COMMD1) during the enrichment for stemness in SAS head and neck squamous-cell carcinoma (HNSCC) cells. The 3'-UTR of COMMD1 mRNA contains microRNA (miR)-205 target site. Parallel studies with HNSCC and NSCLC cells indicated that miR-205 is upregulated upon NF-κB activation and suppresses COMMD1 expression in stemness-enriched cancer cells. COMMD1 negatively regulates the inflammatory responses induced by TLR agonists, IL-1ß, and TNF-α by targeting RelA for degradation. The shRNA-mediated downregulation of COMMD1 in cancer cells enhanced inflammatory response, generating favorable conditions for macrophage recruitment. In addition, genes associated with stemness were also upregulated in these cells, which exhibited increased potential for anchorage-independent growth. Furthermore, COMMD1 downregulation promoted in vivo tumorigenesis and tumor growth, and tumors derived from COMMD1-knockdown cells displayed elevated level of NF-κB activation, increased expression of inflammatory- and stemness-associated genes, and contain expanded population of tumor-associated leukocytes and stemness-enriched cancer cells. These results suggest that COMMD1 downregulation by miR-205 promotes tumor development by modulating a positive feedback loop that amplifies inflammatory- and stemness-associated properties of cancer cells.

Mouse type-2 retinaldehyde dehydrogenase (RALDH2): genomic organization, tissue-dependent expression, chromosome assignment and comparison to other types.

Retinaldehyde dehydrogenase (RALDH) isozymes catalyze the formation of an essential developmental modulator, retinoic acid. We determined the structural organization of mouse type-2 Raldh2 by isolation of overlapping genomic DNA clones from a phage library. The gene consists of 14 exons spanning more than 70 kb of genomic DNA. It was localized to mouse chromosome 6. Northern blot analysis revealed testis-specific expression. The RALDH genes belong to the aldehyde dehydrogenase (ALDH) multi-gene family. Three types of RALDH genes (e.g. human ALDH1/mouse Ahd2/rat RalDH(I), human ALDH11/mouse Raldh2/rat RalDH(II) and human ALDH6) are highly conserved during evolution, sharing about 70% identity at the amino acid level between any two gene types and 90% identity between any two mammalian genes of the same type. Different RALDH types show specific tissue and developmental expression patterns, suggesting (i) a regulatory mechanism of retinoic acid synthesis via different promoters of RALDH genes, and (ii) distinctive biological roles of different isozymes in embryogenesis and organogenesis.

Characterization of the consequence of a novel Glu-380 to Asp mutation by expression of functional P450c21 in Escherichia coli.

P450c21 catalyzes an important step in steroid synthesis. Its deficiency leads to symptoms of steroid imbalance. To obtain enough P450c21 for structure and function studies, we developed a method to express P450c21 in Escherichia coli. The 5'-region of the human P450c21 cDNA was modified to ensure efficient translation and the C terminus of the protein was extended with four His residues for easy purification. Mutant proteins with substitutions at residues 172 and 281 exhibited decreased enzymatic activities similar to those found in mammalian cells. One new mutation changing Glu-380 to Asp (D380) caused 3-fold reduction in enzymatic activity. The amount of apoprotein production detected by immunoblotting and the affinity of the mutant protein towards substrate as measured by Km were normal. The defect lies in the decreased ability of the apoprotein to bind heme, which was measured by CO difference and substrate-binding spectra. The D380 mutant protein had 3-fold reduction in peak heights in both spectra. This reduced heme binding resulted in 3-fold lower enzymatic activity.

The impact of the 1993 AIDS case definition on the completeness and timeliness of AIDS surveillance.

OBJECTIVE: To assess the impact of the 1993 change in the AIDS case definition on the completeness and timeframe of AIDS case reporting in San Francisco. DESIGN: Retrospective review of records: billing records, list of selected diagnostic codes, radiology logs, ophthalmology clinic records, and patient registries at a selection of hospitals, clinics, and physician offices. SETTING: Hospitals, public/community health clinics, and physician offices. MAIN OUTCOME MEASURES: The completeness of reporting and the median reporting delay was calculated for hospitals, clinics, and physician offices. RESULTS: Reporting was 97% complete. Reporting from physician offices was less complete (75%) than from other facilities. The median reporting delay was 1 month and was shorter for persons who met the 1993 AIDS case definition (1 month) than for persons who met the 1987 case definition (3 months). CONCLUSIONS: AIDS case reporting in San Francisco is highly complete but less so for persons diagnosed at physician offices. The 1993 AIDS case definition has resulted in more timely reporting. Health departments should consider efforts to improve reporting from private physician offices and should evaluate the use of laboratory-initiated CD4 reporting.

Spectral analysis of arterial blood pressure and cerebral blood flow velocity during supine rest and orthostasis.

This study evaluates the effect of orthostasis on the low frequency (LF, 0.04 to 0.15 Hz) fluctuations in the blood flow velocity of the middle cerebral artery (MCAFV) in relation to its arterial blood pressure (ABP) equivalent to further define and quantify this relationship in cerebrovascular regulation. Spectral analysis was performed on 22 healthy subjects during supine rest and head-up tilt. The power in the LF range can be used to quantify the LF fluctuations, and four types of LF power data could be obtained for each individual: LF power of supine MCAFV, LF power of supine ABP, LF power of tilt MCAFV, and LF power of tilt ABP. By comparing LF power of MCAFV with LF power of ABP, two power ratios could be generated to describe the flow-pressure relationship during supine rest and head-up tilt, respectively, supine power ratio (LF power of supine MCAFV/ LF power of supine ABP) and tilt power ratio (LF power of tilt MCAFV/ LF power of tilt ABP). In addition, an index for dynamic autoregulation in response to orthostasis can be calculated from these two power ratios (tilt power ratio/supine power ratio). The authors found that this index was dependent on the extent of orthostatic MCAFV changes, and the dependency could be mathematically expressed (r = 0.61, P = .0001), suggesting its involvement in cerebrovascular regulation. Moreover, these data further support the previous observation that the LF fluctuations of MCAFV might result from modulation of its ABP equivalent, and the modulation effect could be quantified as the power ratio (LF power of MCAFV/ LF power of ABP). These observations could be an important step toward further insight into cerebrovascular regulation, which warrants more research in the future.

Transfer function analysis of cerebral hemodynamics in patients with carotid stenosis.

This study evaluates the validity of the transfer function analysis of spontaneous fluctuations of arterial blood pressure (ABP) and blood flow velocity of the middle cerebral artery (MCAFV) as a simple, convenient method to assess human cerebral autoregulation in patients with carotid stenosis. Eighty-three consecutive patients with various degrees of carotid stenosis and 37 healthy controls were enrolled. The carotid stenosis was graded based on the diagnostic criteria of duplex ultrasound. Instantaneous bilateral MCAFV and ABP of all participants were assessed noninvasively using transcranial Doppler sonography and the servocontrolled infrared finger plethysmography, respectively. Spectral analyses of ABP and MCAFV were performed by fast Fourier transform. The fluctuations in ABP as well as in MCAFV were diffracted into three components at specific frequency ranges designated as high-frequency (HF; 0.15 to 0.4 Hz), low-frequency (LF; 0.04 to 0.15 Hz), and very low-frequency (VLF; 0.016 to 0.04 Hz). Cross-spectral analysis was applied to quantify the coherence, transfer phase, and magnitude in individual HF, LF, and VLF components. Transcranial Doppler CO2 vasomotor reactivity was measured with 5% CO2 inhalation. The LF phase angle (r=-0.53, P<0.001); magnitude of VLF (r=-0.29, P=0.002), LF (r=-0.35, P<0.001), and HF (r=-0.47, P<0.001); and CO2 vasomotor reactivity (r=-0.66, P<0.001) were negatively correlated with the severity of stenosis. Patients with unilateral high-grade (greater than 90% stenosis) carotid stenosis demonstrated significant reduction in LF phase angle (P<0.001) and HF magnitude (P=0.018) on the ipsilateral side of the affected vessel compared with their contralateral side. The study also revealed a high sensitivity, specificity, and accuracy using LF phase angle and HF magnitude to detect a high-grade carotid stenosis. A strong correlation existed between the LF phase angle and the CO2 vasomotor reactivity test (r=0.62, P<0.001), and the correlation between the HF magnitude and the CO2 vasomotor reactivity (r=0.44, P<0.001) was statistically significant as well. We conclude that transfer function analysis of spontaneous fluctuations of MCAFV and ABP could be used to identify hemodynamically significant high-grade carotid stenosis with impaired cerebral autoregulation or vasomotor reserve.

Sequencing and expression of the human ALDH8 encoding a new member of the aldehyde dehydrogenase family.

The human aldehyde dehydrogenase gene (ALDH) family is characterized by two major conserved DNA sequences encoding residues which are possibly involved in the catalytic function and the maintenance of the functional conformation of the ALDH enzyme. This property is the basis for synthesizing the degenerate primers to clone several cDNAs of the ALDH isozymes. In this report, we describe the cDNA sequence and the expression of a new member of this family, ALDH8. The human ALDH8 gene was identified during the process of the screening for the human ALDH7 genomic clones. Overlapping ALDH8 cDNA clones were isolated by polymerase chain reaction (PCR) amplification of human salivary gland total RNA or lambda gt11 cDNA library. When the ALDH8 cDNA sequence was aligned with that of the ALDH7 which encodes a polypeptide chain of 468 amino acid (aa) residues, it was found that a termination codon (TGA) is placed in frame at the ALDH8 sequence corresponding to the codon GCG for the seventeenth aa position of the ALDH7. Therefore, the human ALDH8 gene is a potential nonprocessed pseudogene in the ALDH multigene family which has no other pseudogenes reported so far. Alternatively, the ALDH8 gene is a functional gene if the premature stop codon is suppressed, or if the first downstream in-frame ATG serves as the initiator codon. This longest putative open reading frame (ORF) encodes a polypeptide chain of 385 aa residues, includes the two ALDH conserved regions, and demonstrates 86% identity with the corresponding ORF region of the human ALDH7. The expression of the ALDH8 transcripts is restricted to the salivary gland among the human tissues examined.