Engineering anthracycline biosynthesis toward angucyclines

The biosynthesis pathways of two anthracyclines, nogalamycin and aclacinomycin, were directed toward angucyclines by using an angucycline-specific cyclase, pgaF, isolated from a silent antibiotic biosynthesis gene cluster. Addition of pgaF to a gene cassette that harbored the early biosynthesis genes of nogalamycin resulted in the production of two known angucyclinone metabolites, rabelomycin and its precursor, UWM6. Substrate flexibility of pgaF was demonstrated by replacement of the nogalamycin minimal polyketide synthase genes in the gene cassette with the equivalent aclacinomycin genes together with aknE2 and aknF, which specify the unusual propionate starter unit in aclacinomycin biosynthesis. This modification led to the production of a novel angucyclinone, MM2002, in which the expected ethyl side chain was incorporated into the fourth ring.     

Regulation of monocyte migration by amphoterin (HMGB1)

Amphoterin (HMGB1) is a 30-kD heparin-binding protein involved in process extension and migration of cells by a mechanism involving the receptor for advanced glycation end products (RAGE). High levels of amphoterin are released to serum during septic shock. We have studied the expression of amphoterin in monocytes and the role of amphoterin and RAGE in monocyte transendothelial migration. Un-activated monocytes in suspension did not reveal amphoterin on their surface, but adherent monocytes exported amphoterin to the cell surface. Immunohistochemical staining of arterial thrombi in vivo revealed amphoterin in mononuclear cells and in surrounding extracellular matrix. Amphoterin was secreted from phorbol ester and interferon-gamma (IFN-gamma)-activated macrophages, and the secretion was inhibited by blocking the adenosine 5'-triphosphate (ATP)-binding cassette transporter-1, a member of the multidrug resistance protein family. Amphoterin was specifically adhesive for monocytes in peripheral blood leukocyte adhesion assay. Adhesion caused an extensive spreading of cells, which was inhibited by the dominant-negative RAGE receptor (soluble ectodomain of RAGE), and adhesion up-regulated chromogranin expression in monocytes, also suggesting a RAGE-dependent interaction. Monocyte transendothelial migration was efficiently inhibited by anti-amphoterin and anti-RAGE antibodies and by the soluble RAGE. We suggest that amphoterin is an autocrine/paracrine regulator of monocyte invasion through the endothelium.     

Impaired negative chronotropic response to adenosine in patients with inappropriate sinus tachycardia

INTRODUCTION: Adenosine is an endogenous nucleoside that has an important role in the diagnosis and treatment of several cardiac arrhythmias. However, its effects on inappropriate sinus tachycardia (IST) are not well established. METHODS AND RESULTS: In this study, the response to intravenous adenosine (0.1 to 0.15 mg/kg) was studied in 18 patients (age 46+/-15 years) with IST. In a subset of patients (n = 5), the direct effects of adenosine were assessed during pharmacologic beta-adrenergic and cholinergic blockade. Atrial cycle length (ACL) was measured before adenosine injection, at the time of the greatest cycle length prolongation, and during the maximum rebound acceleration of heart rate. Eighteen subjects (age 46+/-11 years) with normal sinus rhythm undergoing clinically indicated electrophysiologic study served as controls. Adenosine did not terminate IST in any patient. The maximum dose of adenosine prolonged the sinus interval significantly, from 780+/-128 msec to 985+/-225 msec (P < 0.001) in the control subjects. In contrast, adenosine caused no significant lengthening of atrial cycle length (527+/-69 msec vs 590+/-148 msec; P = NS) in the patients with IST. Similar difference in the response to adenosine was seen during the pharmacologic autonomic blockade. The reflex increase of the sinus rate (rebound effect) was greater in the control subjects than in the patients with IST (21.2%+/-9.7% vs 8.5%+/-8.8%; P < 0.001). CONCLUSION: The usual negative chronotropic effect of adenosine was impaired in the patients with IST. This may have important diagnostic implications and provide new insight into the mechanism(s) of IST.     

Studies on the Role of Lipid Peroxidation in the Acute Toxicity of TCDD in Rats*

Lipid peroxidation has been shown to be enhanced following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), but its role in TCDD toxicity is unclear. The present study was undertaken to further elucidate the relations between lipid peroxidation and TCDD lethality. A time course and dose-response experiment in Long-Evans (L-E; LD50 ca. 10 μg/kg) and Han/Wistar (H/W; LD50 > 3000 μg/kg) rats showed that hepatic lipid peroxidation, measured as the amount of thiobarbituric acid-reactive substances (TBA-RS), was induced by TCDD dose-dependently in L-E, but not in H/W rats. Hepatic glutathione peroxidase activity was suppressed in much the same manner in both strains. Lipid peroxidation correlated with body weight loss in L-E rats alone. When 500 μg/kg of TCDD was given to L-E rats, lipid peroxidation increased about 3-fold on Day 11 in the liver, while no change was seen in cardiac or renal TBA-RS. The pair-fed controls did not survive the 11-day test period and exhibited gastrointestinal hemorrhages. At 6 days, liver atrophy and elevated (over 2-fold) TBA-RS values were recorded in pair-fed controls but not in their TCDD-treated counterparts. TCDD decreased hepatic glutathione peroxidase activity by almost 50% at 6 days, while pair-feeding was without effect. Liver morphology was different between TCDD-treated and pair-fed rats. Moreover, the livers of TCDD-treated L-E rats contained much higher concentrations of probably peripheral fat-derived fatty acids than did the livers of pair-fed or ad libitum control rats. Restricted feeding over 6 days induced hepatic lipid peroxidation more in H/W than in L-E rats. Endotoxin increased liver TBA levels similarly in both strains having an additive effect with high doses of TCDD in H/W rats. Added as a 0.5% concentration in chow, butylated hydroxyanisole (BHA), but not ethoxyquin, tended to increase survival rate and time in L-E rats exposed to 20 μg/kg of TCDD; at 50 μg/kg the only survivor was again in the BHA group. However, neither antioxidant had any effect on initial body weight loss. It is concluded that lipid peroxidation mainly arises as a secondary phenomenon in TCDD toxicity, is not the cause of the typical histopathological liver lesion, but may contribute to lethality.     

Sex differences in striatal presynaptic dopamine synthesis capacity in healthy subjects.

BACKGROUND: There are sex differences in the clinical features of several neuropsychiatric illnesses associated with dopamine dysfunction. The effects of sex on brain dopaminergic function have been sparsely studied in human subjects using modern imaging techniques. We have previously reported that the apparent affinity of [(11)C]raclopride for striatal D(2) dopamine receptors in vivo is lower in women than in men, whereas D(2) receptor density is not different. This finding indirectly suggests that women have a higher synaptic concentration of dopamine in the striatum. We explored further the basis of this phenomenon in an independent study and hypothesized that striatal presynaptic dopamine synthesis capacity would also be elevated in women. METHODS: A total of 23 healthy men and 12 healthy women (age range 20-60 years) were studied using positron emission tomography and [(18)F]fluorodopa. RESULTS: Women had significantly higher striatal [(18)F]fluorodopa uptake (Ki values) than men. The difference was more marked in the caudate (+26%) than in the putamen (+12%). In addition, there was a negative correlation between striatal [(18)F]fluorodopa Ki values and age in men but not in women. CONCLUSIONS: The results further substantiate sex differences in striatal dopaminergic function in humans. This finding may be associated with sex differences in vulnerability and clinical course of neuropsychiatric disorders with dopaminergic dysregulation, e.g., schizophrenia, alcohol dependence, and Parkinson's disease.     

Pigmentary retinopathy in patients with the MELAS mutation 3243A→G in mitochondrial DNA

BACKGROUND: Our objective was to determine the penetrance of retinal pigment epithelium (RPE) abnormalities and other ophthalmologic manifestations in patients with the 3243A-->G mutation in mitochondrial DNA. METHODS: Adult members in two generations were examined from a population-based cohort of 13 pedigrees with 3243A-->G. Twenty-six patients underwent a thorough ophthalmological examination. A chart review was carried out on an additional 44 patients. RESULTS: Paramacular RPE atrophy and areas of hyperpigmentation were found in 10 patients (38%; 95% confidence interval 20-59%). Electroretinography was normal in only one of the eight patients tested, whereas dark adaptation was abnormal in two. RPE abnormalities were associated with more severe clinical phenotypes and higher degrees of 3243A-->G mutation heteroplasmy in muscle. Ten patients had diabetes mellitus, nine of whom had also RPE abnormalities. This finding, however, reflected the severity of the phenotype, and diabetic retinopathy was confidently diagnosed in only two patients. External ophthalmoplegia was detected in occasional patients. CONCLUSION: RPE abnormalities were found in this population-based cohort at a frequency that was lower than that reported earlier. RPE abnormalities were associated with more severe phenotypes, suggesting that they are expressed in patients with syndromic features. RPE abnormalities and diabetes mellitus co-occurred frequently, but diabetic retinopathy was not common.     

Endogenous Histamine in Cultured Bovine Adrenal Chromaffin Cells

Histamine releases catecholamines and opioids in primary cultured bovine adrenal medullary (BAM) chromaffin cells. We have studied whether histamine is synthesized and localized in BAM cells, and whether it can be released upon activation with secretagogues. In BAM cells histamine is immunohistochemically co-localized with tyrosine hydroxylase in 45 ± 8% of all cells. Only histamine immunoreactivity was observed in 8 ± 2% of all BAM cells. No mast-cell-like cells were observed in our system. Histamine can be released from BAM cells by high potassium (56 mM K⁺) in a calcium-dependent manner. Compound 48/80 did not release histamine from BAM cells but nicotine caused a dose-dependent liberation of the amine. Cultured BAM cells have histidine decarboxylase activity which is inhibited by α-fluoromethylhistidine. These results indicate that endogenous histamine is synthesized, stored and released in BAM chromaffin cells in vitro.     

The prognosis of demented patients — one-year follow-up study of a population sample

Functional decline and mortality after 1-year follow-up of 93 demented elderly subjects from a random population sample (N =795) were studied in Helsinki, Finland. Seventeen (18%) of the demented patients died as compared to only 5% of the non-demented subjects. The age-adjusted risk ratio for 1-year mortality of demented patients was 3.2 (95% confidence interval 1.8–5.6). Forty-four per cent (26/59) of the surviving patients who were not bedridden and incontinent at the very beginning of the study suffered further impairment in the following functions: institutionalization (6/21), losing the ability to walk (5/57) or beginning of incontinence (17/30). Sixteen (21%) surviving subjects were already bedbound, incontinent and institutionalized at the beginning of the study. Age, sex or the degree of dementia did not correlate to mortality or functional decline. The number of patients who were institutionalized during the follow-up was small. The appearance of incontinence and other recorded risk factors (age, degree of dementia or motility) did not seem to predispose to institutionalization. Subjective factors, such as caretaker's burden, probably have a great influence on the ending of home care.