Faculty Bibliography

Ouabain is a well known inhibitor of the Na+ pump in all mammalian cells. We have demonstrated that ouabain at concentrations below those which inhibit the pump, i.e. 0.1 nM and 1.0 nM, induce proliferation of saphenous vein smooth muscle cells as measured by bromodeoxyuridine (BrdU) uptake. Ouabain at these low concentrations also activated MAPK. Proliferating concentrations of the drug did not increase levels of Ca(i)2+, suggesting no effect of this ion in the process. In addition, incubation of the cells in low levels of K+, which has been shown to inhibit the pump, had no effect on proliferation. These data show that low concentrations of ouabain that do not inhibit the Na+ pump can activate proliferation of vascular smooth muscle cells, suggesting that the pump complex may act as a transducing receptor.

The hypothesis of this study is that the sodium pump complex acts as an intracellular signal-transducing molecule in canine vascular smooth muscle cells through its interaction with other membrane and cytoskeletal proteins. We have demonstrated that 1 nm ouabain induced transactivation of the epidermal growth factor receptor (EGFR), resulting in increased proliferation and bromodeoxyuridine (BrdUrd) uptake. Immunoprecipitation and Western blotting showed that the EGFR and Src were phosphorylated within 5 min of 10(-9) m ouabain stimulation. Both ouabain-induced DNA synthesis (BrdUrd uptake) and MAPK42/44 phosphorylation were inhibited by the Src inhibitor PP2, the EGFR kinase inhibitor AG1478, the tyrosine kinase inhibitor genistein, and the MEK1 inhibitor PD98059. Ouabain concentrations higher than 1 nm had little or no stimulating effect on proliferation or BrdUrd uptake but did minimally activate ERK1/2. Thus, low concentrations of ouabain, which do not inhibit the sodium pump sufficiently to perturb the resting cellular ionic milieu, initiate a transactivational signaling cascade leading to vascular smooth muscle cell proliferation.

Bovine milk will eventually spoil at refrigeration temperatures, but endogenous or exogenous pathogenic or spoilage bacteria in human milk stored for delayed feeding will die. We investigated the mechanism for these antibacterial properties and their response to high-tempertature, short-time (HTST, 72 degrees C-75 degrees C, 15 sec) and low-temperature long-time (LTLT, 65 degrees C, 30 min) pasteurization. Nonpathogenic Listeria innocua (10(6) cfu/mL) was inoculated into raw and processed bovine and human milk; bacterial plate counts twice weekly determined antibacterial activities. Up to 99% of L. innocua were killed and further growth was inhibited in raw and pasteurized human milk for at least 60 days at 4 degrees C. Reactive IgA antibodies against Listeria antigens were demonstrated by enzyme immunoassay in some human milk samples; sIgA activity against Escherichia coli O antigens was significantly decreased by heat treatments (raw, 1.8; HTST, 1.1; LTLT, 1.3 activity units). Adding human lactoferrin (0.5-20 mg/mL) to the Listeria inoculum (approximately 10(7) cfu/mL) in 1% peptone water did not inhibit bacterial growth.

Leber's hereditary optic neuropathy (LHON) is characterized by maternally transmitted, bilateral, central vision loss in young adults. It is caused by mutations in the mitochondrial DNA (mtDNA) encoded genes that contribute polypeptides to NADH dehydrogenase or complex I. Four mtDNA variants, the nucleotide pair (np) 3460A, 11778A, 14484C, and 14459A mutations, are known as "primary" LHON mutations and are found in most, but not all, of the LHON families reported to date. Here, we report the extensive genetic and biochemical analysis of five Russian families from the Novosibirsk region of Siberia manifesting maternally transmitted optic atrophy consistent with LHON. Three of the five families harbor known LHON primary mutations. Complete sequence analysis of proband mtDNA in the other two families has revealed novel complex I mutations at nps 3635A and 4640C, respectively. These mutations are homoplasmic and have not been reported in the literature. Biochemical analysis of complex I in patient lymphoblasts and transmitochondrial cybrids demonstrated a respiration defect with complex-I-linked substrates, although the specific activity of complex I was not reduced. Overall, our data suggests that the spectrum of mtDNA mutations associated with LHON in Russia is similar to that in Europe and North America and that the np 3635A and 4640C mutations may be additional mtDNA complex I mutations contributing to LHON expression.

The black blow fly, Phormia regina (Meigen) is a primary species commonly utilized to indicate a postmortem interval, or more appropriately a "time since colonization". Due to the importance of this species as a secondary myiasis producer in livestock operations, and more recently as a time since death indicator in the field of forensic entomology, a considerable amount of data on its growth and development has been generated. However, the developmental time as reported by these studies varies greatly, and current more detailed data is needed for use in medicocriminal entomology. Hourly developmental data is presented under constant temperatures of 10, 15, 20, 25, 30, 35 and 40 degrees C, and cyclic temperatures of 10-15, 15-25, 25-35 and 35-45 degrees C. This study is in agreement with the results reported by Kamal [Comparative study of thirteen species of sarcosaprophagous Calliphoridae and Sarcophagidae (Diptera). I. Bionomics, Ann. Entomol. Soc. Am. 51 (1958) 261] and Melvin [Incubation period of eggs of certain musciod flies at different constant temperatures, Ann. Entomol. Soc. Am. 27 (1934) 406] only at temperatures of 25 degrees C and below. Bishopp [Flies which cause myiasis in man and animals: some aspects of the problem, J. Econ. Entomol. 8 (1915) 317] reported a shorter developmental duration for larval stages than what was produced with our laboratory rearings.

The whey protein alpha-lactalbumin, derived from human milk, has been shown to inhibit proliferation of mammary epithelial cells and rat kidney cells. We have shown that bovine alpha-lactalbumin also has antiproliferative effects in human colon adenocarcinoma cell lines. During a 5-day dose-dependent growth study, bovine alpha-lactalbumin was added to Caco-2 or HT-29 monolayers in amounts from 5 to 35 microg/mL. Low concentrations of alpha-lactalbumin (10-25 microg/mL) stimulated growth during the first 3 to 4 days. After growing for 4 days, proliferation ceased and viable cell numbers decreased dramatically in the alpha-lactalbumin-treated cultures, suggesting a delayed initiation of apoptosis. This experiment demonstrates the acute bioactive effects of small concentrations of alpha-lactalbumin, compared with the high concentrations of other proteins in the media. These results suggest that alpha-lactalbumin in milk may promote health by inhibiting growth of potential cancer cells. Further studies will identify the role of calcium in the bioactivity of alpha-lactalbumin.

OBJECTIVE: To determine any differences in cerebral perfusion pressure in patients with chronic hypertension compared with those with chronic hypertension and superimposed pre-eclampsia. DESIGN: A prospective observational study. SETTING: University hospital clinic and labour and delivery suite. PARTICIPANTS: Fifteen women with chronic hypertension and 15 with superimposed pre-eclampsia. METHODS: Transcranial Doppler ultrasound was used to measure blood velocity in the middle cerebral arteries of the patients. Systemic blood pressure in the brachial artery was measured simultaneously. Middle cerebral artery. resistance index, pulsatility index, and cerebral perfusion pressure were calculated and plotted on the same axes as data from normal pregnant women. Cerebral perfusion pressure values outside of the 5th and 95th centiles were regarded as abnormal. Cerebral perfusion pressure data from the chronic hypertension and superimposed pre-eclampsia groups were also expressed in terms of the number of normative standard deviations from the mean value for normal pregnancy (Multiples of the Standard Deviation: MOS). All studies were conducted before labour, under similar conditions, and before volume expansion or treatment. Statistical analysis was by Student's t test and Fisher's exact test as appropriate with significance set at a two-tailed P<0.05. RESULTS: Patient demographics and blood pressure were not significantly different between the two groups. The resistance index and pulsatility index were not significantly different (neither absolute nor multiples of the standard deviation values). The absolute cerebral perfusion pressure was significantly higher in the patients with superimposed pre-eclampsia. The group of women with superimposed pre-eclampsia had a significantly higher mean value of cerebral perfusion pressure measured as multiples of the standard deviation from the mean value for normal pregnancy, despite there being no blood pressure difference. CONCLUSIONS: Superimposed pre-eclampsia is associated with significantly higher cerebral perfusion pressure measurements compared with women with uncomplicated chronic hypertension. This is not directly related to a higher blood pressure. The difference in cerebral perfusion pressure may be used to speculate upon the pathophysiology of the increased risk for eclampsia seen in patients with superimposed pre-eclampsia.

The Na(+) pump and its regulation is important for maintaining membrane potential and transmembrane Na(+) gradient in all mammalian cells and thus is essential for cell survival and function. Vascular smooth muscle cells (VSMC) have a relatively low number of pump sites on their membrane compared with other cells. We wished to determine the mechanisms for regulating the number of pump sites in these cells. We used canine saphenous vein VSMC cultured in 10% serum and passaged one time. These cells were subcultured in 5% serum media with low K(+) (1 mM vs. control of 5 mM), and their pump expression was assessed. These VSMC upregulated their pump sites as early as 4 h after treatment (measured by [(3)H]ouabain binding). At this early time point, there was no detectable increase in protein expression of either alpha(1)- or beta(1)-subunits of the pump shown by Western blots. When the cells were treated with the phosphoinositide 3-kinase (PI-3-K) inhibitor LY-294002 (which is known to inhibit cytoplasmic transport processes) in low-K(+) media, the pump site upregulation was inhibited. These data suggest that the low-K(+)-induced upregulation of Na(+) pump number can occur by translocation of preformed pumps from intracellular stores.

Duck embryo was studied as a model for assessing the effects of microbeam radiation therapy (MRT) on the human infant brain. Because of the high risk of radiation-induced disruption of the developmental process in the immature brain, conventional wide-beam radiotherapy of brain tumors is seldom carried out in infants under the age of three. Other types of treatment for pediatric brain tumors are frequently ineffective. Recent findings from studies in Grenoble on the brain of suckling rats indicate that MRT could be of benefit for the treatment of early childhood tumors. In our studies, duck embryos were irradiated at 3-4 days prior to hatching. Irradiation was carried out using a single exposure of synchrotron-generated X-rays, either in the form of parallel microplanar beams (microbeams), or as non-segmented broad beam. The individual microplanar beams had a width of 27 microm and height of 11 mm, and a center-to-center spacing of 100 microm. Doses to the exposed areas of embryo brain were 40, 80, 160 and 450 Gy (in-slice dose) for the microbeam, and 6, 12 and 18 Gy for the broad beam. The biological end point employed in the study was ataxia. This neurological symptom of radiation damage to the brain developed within 75 days of hatching. Histopathological analysis of brain tissue did not reveal any radiation induced lesions for microbeam doses of 40-160 Gy (in-slice), although some incidences of ataxia were observed in that dose group. However, severe brain lesions did occur in animals in the 450 Gy microbeam dose groups, and mild lesions in the 18 Gy broad beam dose group. These results indicate that embryonic duck brain has an appreciably higher tolerance to the microbeam modality, as compared to the broad beam modality. When the microbeam dose was normalized to the full volume of the irradiated tissue. i.e., the dose averaged over microbeams and the space between the microbeams, brain tolerance was estimated to be about three times higher to microbeam irradiation as compared with broad beam irradiation.

Four mitochondrial DNA (mtDNA) mutations at nps 3460, 11778, 14484, and 14459 account for roughly 90% of cases of Leber hereditary optic neuropathy (LHON) and are designated as "primary" LHON mutations since they act as major predisposition factors for LHON. Although each primary mutation can arise independently on different mtDNA backgrounds during human evolution, they characteristically do not co-occur in LHON patients. We report here a family with the simultaneous occurrence of the 11778A and 14484C mutations. Neuro-ophthalmological examination of the proband, a nine-year-old Caucasian female, revealed the bilateral optic atrophy, central scotomas, and reduced visual acuity typical of LHON. Her mother had normal appearing optic discs and is today visually asymptomatic. Analysis of the proband blood mtDNA revealed that she harbored both the 11778A (heteroplasmic, 94% mutant) and the 14484C (homoplasmic mutant) mutation. This genotype was maintained in proband lymphoblasts and transmitochondrial cybrids. The mother also had both mutations, with the 14484C mutation homoplasmic in all cell types examined. However, only 31% of her blood mtDNAs carried the 11778 mutation, which segregated to essentially 100% wild-type in lymphoblast and cybrid mtDNA. Complex I-linked respiration and specific enzyme activity were consistently lowest in proband lymphoblast and cybrid mitochondria compared to those from the mother, 11778A patients, 14484C patients, or controls, thus demonstrating both a deleterious synergistic interaction between the 11778A and 14484C mutations and the magnitude of 11778A-associated complex I dysfunction. Remarkably, spontaneous vision recovery occurred in the proband, highlighting the complexities encountered when associating mtDNA genotype and complex I function with LHON expression.