Heat-related death and mental illness during the 1999 Cincinnati heat wave.

During a 1999 heat wave in Cincinnati, Ohio, the Hamilton County Coroner reported 18 heat-related deaths. The Centers for Disease Control and Prevention and the Cincinnati Department of Health conducted a case-control study using surrogate case information and first-person control information to identify risk factors for mortality during the heat wave. Surrogate data were supplemented by systematic death scene investigation reports and comprehensive toxicologic screens, important sources of data that are routinely collected by the Hamilton County Coroner's Office. The study included 17 case subjects and 34 controls from the decedents' neighborhood. Among 17 case subjects, 8 (47.1%) had mental illness (odds ratio [OR], 14.0; 95% confidence interval [CI], 1.8-633). There was a suggestion of an interaction between age and mental health. A working air-conditioner was the strongest protective factor (OR, 0.03; 95% CI, 0-0.2). Toxicologic screening indicated that case subjects with reported mental illness and a prescription for psychotropic drugs may not have been medication compliant. Three decedents lived in group homes for people with mental illness, indicating that opportunities for prevention may have been missed. Systematic death investigations, including toxicologic screening, provide valuable information about the circumstances of heat-related death, particularly the role of medication compliance as a risk factor. Prevention programs during heat waves should target people with mental illness, especially those who take psychotropic medication.

Apolipoprotein E deficiency in mice: gene replacement and prevention of atherosclerosis using adenovirus vectors.

Apolipoprotein E (apoE)-deficient mice develop marked hyperlipidemia as well as atherosclerosis and thus are an excellent animal model for evaluating the potential for gene therapy in human genetic dyslipoproteinemias. Recombinant adenovirus containing either human apoE (rAdv.apoE) or the reporter gene luciferase (rAdv.luc) were generated and infused intravenously in apoE-deficient mice with preinfusion plasma total cholesterol of 644 +/- 149 mg/dl an cholesterol rich VLDL/IDL. After a single infusion of rAdv.apoE, plasma concentrations of human apoE ranging from 1.5 to 650 mg/dl were achieved. Adenovirus-mediated apoE replacement resulted in normalization of the lipid and lipoprotein profile with markedly decreased total cholesterol (103 +/- 18mg/dl), VLDL, IDL, and LDL, as well as increased HDL. Measurement of aortic atherosclerosis 1 mo after adenoviral infusion demonstrated a marked reduction in the mean lesion area of mice infused with rAdv.apoE (58 +/- 8 x 10(3) microns2) when compared with control mice infused with rAdv.luc (161 +/- 10 x 10(3) microns2; P < 0.0001). Thus, apoE expression for 4 wk was sufficient to markedly reduce atherosclerosis, demonstrating the feasibility of gene therapy for correction of genetic hyperlipidemias resulting in atherosclerosis. The combined use of adenovirus vectors and the apoE-deficient mouse represents a new in vivo approach that will permit rapid screening of candidate genes for the prevention of atherosclerosis.

ApoC-IIParis2: a premature termination mutation in the signal peptide of apoC-II resulting in the familial chylomicronemia syndrome.

The chemical mismatch method has been utilized to screen for mutations in the apoC-II gene of a patient with familial chylomicronemia and apoC-II deficiency. Cleavage of heteroduplexes formed between normal and patient DNA strands with hydroxylamine and osmium tetroxide readily localized a mutation near base 2660 of the mutant apoC-II. Sequence analysis of PCR amplified patient DNA in the mismatched region localized by this method identified the substitution of a thymidine (T) for a cytosine (C) at base 2668 in exon 2 of the patient's gene within a CpG dinucleotide. The C to T transition in the apoC-IIParis2 gene leads to the introduction of a premature termination codon (TGA) at a position corresponding to amino acid-19 of the signal peptide of apoC-II and the formation of a new Nla III restriction enzyme site absent in the normal apoC-II gene. Consistent with the history of consanguinity in this kindred, amplification of DNA isolated from the proband's parents by the polymerase chain reaction and digestion with Nla III established that the proband is a true homozygote for this genetic defect. Analysis of the patient's plasma by two-dimensional gel electrophoresis and immunoblotting failed to detect any plasma apoC-II. Thus, we have identified a novel mutation in the apoC-II gene of a patient with apoC-II deficiency from a Paris kindred presenting with severe hypertriglyceridemia and chylomicronemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional regulation of the human lipoprotein lipase gene in 3T3-L1 adipocytes.

Lipoprotein lipase (LPL), a key enzyme in normal lipoprotein metabolism, has a complex pattern of regulation and tissue-specific expression. Several potential binding sites for transcription factors, including the recognition sequences for CCAAT/enhancer-binding protein and octamer-binding proteins (Oct) have been described in the 5'-flanking region of the human LPL gene. To identify elements which regulate the expression of LPL in adipocytes, plasmids containing deletion mutants of the 5'-LPL promoter region and the luciferase reporter gene were transfected in 3T3-L1 adipocytes. Deletions at -724, -565, -461, -368, -232, -167, -92, -35, and -17 relative to the transcriptional start site modified transcription from 100 to 162, 194, 185, 128, 63, 53, 29, and 0%, respectively, indicating the presence of negative (-724 to -565) and positive (-368 to -35) cis-acting regulatory elements. Transfection of HepG2 cells, which do not synthesize LPL, with the same constructs resulted in a similar pattern of expression for the majority of the deletions. However, deletions between -724 and -368 base pairs resulted in a 75-100% increase in transcription in 3T3 adipocytes but not in HepG2 cells, indicating the presence of tissue-specific regulatory element(s) in this region. An important regulatory element affecting LPL transcription in adipocytes was identified by gel mobility shift assays and DNase I footprint analysis. Using these techniques, a nuclear protein(s) in 3T3-L1 adipocytes was shown to bind specifically to a fragment which included the proximal octamer recognition site (from -46 to -39) present in the LPL promoter. The DNA-protein complex comigrates with an electrophoretic band containing the Oct-1-DNA complex in BJA-B nuclear extracts and the DNA-protein complex was selectively competed only by DNA fragments containing the octamer sequence. Preincubation of 3T3-L1 nuclear extracts with an antibody directed against the POU domain of Oct-1 inhibited the formation of the DNA-protein complex. Deletion of the proximal octanucleotide motif from the plasmid containing the -461 fragment of the LPL promoter, resulted in a 79 and 76% decrease in the level of expression in transfected 3T3-L1 adipocytes and HepG2 hepatocytes, respectively. These combined results have established that the expression of LPL in adipocytes is modulated by multiple positive and negative regulatory elements within the 5'-flanking region of the LPL gene. A proximal octamer binding sequence which specifically interacts with a nuclear protein(s) that exhibits the characteristics of Oct-1 has been identified.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunologic selection of simian virus 40 (SV40) T-antigen-negative tumor cells which arise by excision of early SV40 DNA.

A clonal line of highly oncogenic spontaneously transformed mouse cells (104C) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned (106CSC). This 106CSC cell line expressed T antigen and transplantation antigen but was about 100 times less tumorigenic than the 104C parent. When 10(5) 106CSC cells were injected into immunocompetent syngeneic mice, tumors were produced. From such tumors, cell lines were established in culture, all of which were consistently negative for T antigen. We found previously by solution DNA hybridization methods that the tumor cells were depleted in the early region of SV40 DNA which codes for the T antigen. We postulated that this loss occurs through a DNA rearrangement of unknown mechanism in one or a few 106CSC cells and that the tumors are then produced from such a cell or cells, whereas all the T-antigen-positive 106CSC cells are rejected by immunologic means. In this investigation we showed by the DNA transfer method using appropriately selected SV40 DNA probes that indeed the tumor cell clone (130CSCT) we selected to investigate came from one 106CSC cell in which the T-antigen-coding SV40 DNA sequences (but not all the early SV40 DNA sequences) were lost by an excision and recombination mechanism. We also showed that the 130CSCT cells, which are highly tumorigenic, could again be transformed by SV40 and that the resulting T-antigen-positive cloned derivative cells became much less tumorigenic (approximately 10(5)-fold), apparently again because of immunologic recognition and rejection. Indeed, when 10(7) T-antigen-positive cloned cells were injected, all the T-antigen-positive cells were rejected and the tumor was produced again from one or more T-antigen-negative cells. Thus, a one-step in vivo transplantation experiment allowed a selection (for tumorigenicity and against the SV40 T antigen) of a mutant mammalian cell with a DNA deletion at a definable site.

Disseminated coccidioidomycosis in a heart transplant recipient.

A cardiac transplant patient developed disseminated coccidioidomycosis shortly after transplantation and institution of immunosuppressive therapy. The patient was maintained on intravenous and intrathecal amphotericin B for 19 months, but when therapy was discontinued, the disease relapsed and he died. At autopsy the cardiac allograft was without signs of rejection, but the patient had coccidioidomycotic lesions in multiple organs. There is an increasing number of reports of disseminated coccidioidomycosis in immunocompromised patients, especially those who receive steroids or immunosuppressive therapy. Coccidioidomycosis may represent a severe complication in the transplant patient.

A spectrophotometric study of the reaction of boro-hydride with trinitrophenyl derivatives of amino acids and proteins.

The absorption spectra of trinitrophenyl derivatives of poly(L-lysine) and L-asparaginase undergo irreversible changes in the presence of KBH4. The spectra of trinitrophenyl derivatives of N-acetyl-L-lysine and N-acetyl-L-cysteine are also affected by the addition of the reducing agent. A broad absorption band with a maximum at 426 nm appears in the presence of low concentrations of borohydride with a concomitant decrease in absorbance of the 346 nm band which is characteristic of 1-substituted 2,4,6-trinitrophenyl compounds. In the presence of higher concentrations of KBH4 the long wavelength band becomes less broad as the maximum is shifted to 410 nm and the 346 nm band completely disappears. Similar spectral changes were observed in the presence of Na2SO3 although these were reversible upon removal of the sulfite by dialysis. Based on the spectral similarities with sulfite and hydroxide adducts, we suggest that the 426 nm maximum represents a 1:1 adduct formed between the trinitrophenyl moiety and a hydride ion while the band at 410 nm is assigned to the 1:2 adduct.

The effect of trinitrophenylation on subunit interactions in L-asparaginase.

L-Asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) from Escherichia coli B was modified by treatment with 2,4,6-trinitrobenzene-1-sulfonic acid at pH 7.5. The introduction of 13 trinitrophenyl groups into one mol of the tetrameric enzyme (TNP 13-asparaginase) results in a loss of 67% of the catalytic activity while the presence of 20 groups (TNP 20-asparaginase) reduces the enzymatic activity by 88%. The modified proteins are homogeneous as judged by disc gel electrophoresis and by the monodisperse boundary in the analytical ultracentrifuge having a sedimentation coefficient of 7.2 S. The rate of dissociation of the TNP 13-asparaginase is twice as fast and TNP 20-asparaginase three times as fast as that of unmodified asparaginase in 4 M urea. Trinitrophenylated subunits in 8 M urea can reassociate into the tetramer after removal of urea by dialysis or by dilution. hybridization of unmodified and TNP subunits indicates that that trinitrophenyl derivatives qualify as suitable variants for studying subunit interactions in oligomeric proteins.