Ida W. Smoak

Influence of Pregnancy on the Pathogenesis of Listeriosis in Mice Inoculated Intragastrically

ABSTRACT Pregnancy increases the risk of listeriosis, a systemic disease caused by Listeria monocytogenes. However, there is incomplete agreement on the reasons for this increased risk. We examined two features of listeriosis in gravid and nongravid female mice following intragastric (gavage) inoculation, namely, (i) disease severity (measured by lethality) and (ii) listerial infectivity (measured by liver and spleen colonization levels up to 120 h postinoculation). Two listerial strains of differing serotype (1/2a and 4nonb) were initially employed. Neither strain produced a lethal infection in nonpregnant female mice (dose range, 106 to 109 CFU/mouse), and only the 4nonb strain produced lethalities in pregnant mice (dose range, 106 to 108 CFU/mouse). The 4nonb strain also produced a higher level of liver and spleen colonization than the 1/2a strain following gavage administration. (The two strains showed similar levels of colonization if parenterally administered.) Both strains were equally capable of binding to and forming plaques upon cultured mouse enterocytes. The ability of the 4nonb strain to produce a lethal infection in pregnant animals did not correlate with an increased incidence or level of liver and spleen colonization over that in nonpregnant females. However, the lethality rate did correlate well with the rate at which embryos and their surrounding decidual covering became infected, suggesting that intrauterine infection could be responsible for the increased disease severity in the gravid females.

Hyperglycemia-induced TGFβ and fibronectin expression in embryonic mouse heart

Cardiovascular defects are common in diabetic offspring, but their etiology and pathogenesis are poorly understood. Extracellular matrix accumulates in adult tissues in response to hyperglycemia, and transforming growth factor‐beta1 (TGFβ1) likely mediates this effect. The objective of this study was to characterize TGFβ expression in the organogenesis‐stage mouse heart and to evaluate TGFβ and fibronectin expression in embryonic mouse heart exposed to hyperglycemia. Prominent TGFβ1, and minimal TGFβ2 or TGFβ3, protein expression was demonstrated in embryonic day (E) 9.5–E13.5 hearts. Hyperglycemia for 24 hr produced significantly increased fibronectin, slightly increased TGFβ1, and unchanged TGFβ2 or TGFβ3, by immunohistochemistry. Increased TGFβ1 was demonstrated by enzyme‐linked immunosorbent assay in embryonic fluid and isolated hearts after hyperglycemia for 24 hr, but not 48 hr. Hyperglycemia increased fibronectin protein and mRNA expression in embryonic hearts after 24 hr, and pericardial injection of TGFβ1 also increased fibronectin mRNA in the embryonic heart. It is proposed that TGFβ1 and fibronectin may play a role in diabetes‐induced cardiac dysmorphogenesis. Developmental Dynamics 231:179–189, 2004.

Hexokinase I expression and activity in embryonic mouse heart during early and late organogenesis.

Abstract Hexokinase (HK) catalyzes the first step in glucose metabolism, that is, the conversion of glucose to glucose-6-phosphate (G6P). Four HK isoforms have been identified, of which HK-I is predominant in embryonic and fetal tissues. HK-I has been studied in preimplantation embryos and in fetal stages, but little is known about its activity or expression in the early postimplantation embryo. We evaluated HK-I expression, HK-I activity, and glycolytic metabolism in the embryonic mouse heart during early [gestational day (gd) 9.5] and late (gd 13.5) organogenesis. Immunohistochemistry demonstrated that HK-I is localized mainly in the heart at both stages, with stronger expression on gd 13.5. Densitometry after SDS-PAGE/western analysis confirmed higher immunodetectable HK-I protein levels in hearts on gd 13.5 vs gd 9.5. By contrast, RT-PCR demonstrated higher HK-I mRNA expression on gd 9.5 vs gd 13.5. Similarly, cardiac HK-I activity (conversion of glucose to G6P) and glycolysis (conversion of glucose to lactate) were higher on gd 9.5 than on gd 13.5. These results suggest a complex regulation of HK-I expression and activity in the embryonic heart during organogenesis, involving a change in the intrinsic activity of the enzyme with development. HK-I appears to play an important role in glucose metabolism during this critical stage of cardiogenesis.

Expression of glucose-regulate proteins (GRP78 and GRP94) in hearts and fore-limb buds of mouse embryos exposed to hypoglycemia in vitro

Hypoglycemia, the classic inducer of glucose-related protein (GRP) synthesis, is dysmorphogenic in rodent embryos and detrimentally affects the heart. This study compares GRP induction in a target vs non-target tissue by evaluating GRP expression in hearts and fore-limb buds of mouse embryos following exposure to hypoglycemia in vitro. Gestational day 9.5 embryos were exposed to 2, 6, and 24 h of either mild (80 mg/dl glucose) or severe (40 mg/dl glucose) hypoglycemia using the method of whole-embryo culture. GRP78 increased in a dose- and time-dependent fashion in embryonic hearts exposed to either 40 mg/dl or 80 mg/dl glucose, whereas GRP94 levels increased in hearts only after 24 h of hypoglycemia. In contrast to the heart, GRP induction in fore-limb buds occurred only with GRP78 following the most severe level and duration of hypoglycemia. RT-PCR analysis demonstrated an elevation in GRP78 and GRP94 message levels in embryonic hearts following severe hypoglycemia. However, mRNA levels did not increase in response to mild hypoglycemia. Overall, these data demonstrate the preferential induction of GRPs in the heart as compared to fore-limb buds in mouse embryos exposed to hypoglycemia. Increases in GRP protein levels may be a more reliable biomarker of stress than message levels. However, both tissues and methods should be examined for enhanced biomarker sensitivity.

Determination of chlorobutanol in mouse serum, urine and embryos by capillary gas chromatography with electron capture detection.

A sensitive and specific method for the determination of chlorobutanol (1,1,1-trichloro-2-methyl-2-propanol) in mouse serum, urine, and embryos by capillary gas chromatography with electron capture detection is described. For sample preparation n-hexane was used to extract chlorobutanol and the internal standard 2,2,2-trichloroethanol (TCE) from each matrix. Following extraction chromatographic separation of the samples was achieved with a fused-silica capillary column (30 m x 0.25 mm I.D., 0.25 micron film thickness). The method as described has the required sensitivity to quantitate chlorobutanol in individual embryos following administration of a single oral dose of the drug to a pregnant mouse. The limit of detection was 1 pg on column and the detector response was linear from 1 to 100 micrograms/ml for serum, 0.2 to 20 micrograms/ml for urine, and 1 to 10 ng/embryo.