Molecular cloning and cultivar specific expression of MAP kinases from Capsicum annuum.

Two MAP kinases, MK1 and MK2, were cloned from Capsicum annuum (pepper) cv. Subicho using a parsley MAP kinase gene as a heterologous probe. MK1 and MK2 encode stress-inducible protein kinases that can contribute to the response to wounding, UV-C, and cold. MK1 has a 92% amino acid identity with WIPK of tobacco. It was transcriptionally induced in response to wounding. In contrast, no detectable MK1 transcript was found in unwounded leaves of pepper. MK2 has a high level of amino acid homology to MAP kinases, such as NTF4 and SIPK and was constitutively expressed in all tissues. Both MK transcripts were downregulated by UV-C treatment. Each MK protein activation was independently wound-inducible in a cultivar dependent manner. MK1 is phosphorylated in cv. Pungchon but not cv. Subicho; whereas, the MK2 protein activation by wounding is restricted to cv. Subicho. In addition, de novo synthesis of the MK1 protein and tyrosine phosphorylation was rapidly and transiently induced in cv. Pungchon by wounding. In contrast, it is highly unlikely that the MK1 protein is produced in cv. Subicho, even though there is an abundant expression of MK1 mRNA after wounding in this cultivar. In Escherichia coli, which overexpresses MK1, autophosphorylation is observed at conserved threonine and tyrosine phosphorylation sites.

The expression of Bok is regulated by serum in HC11 mammary epithelial cells.

Epithelial cells within the mammary gland undergo apoptosis during weaning. To determine the expression of Bok mRNA (a member of the pro-apoptotic Bcl-2 family) in the mammary gland and its regulation, we examined the expression of the Bok transcript in the mouse mammary gland and HC11 mammary epithelial cells in culture through RT-PCR. The Bok mRNA expression was found in the mammary gland. The expression of the Bok mRNA level was induced through serum starvation and overexpression of Bok induced apoptosis in HC11 cells in culture. These results indicate that the expression of Bok mRNA in the mammary gland is regulated through serum starvation. It also may be related to the mammary involution.

Matrix attachment region sequences enhanced the expression frequency of a whey acidic protein/human lactoferrin fusion gene in the mammary gland of transgenic mice.

To elevate the expression frequency of transgenes in transgenic mice, the chicken lysozyme matrix attachment region (MAR) sequence was used by combining it with a transgene. The whey acidic protein (WAP) promoter/human lactoferrin (hLF) cDNA fusion transgene (pWL) was connected to the chicken lysozyme MAR sequence at its 5'-end (pMWL). While only two of three mice became transgenic from the pWL vector expressed hLF, all seven mice from the pMWL vector expressed the transgene in their lactating mammary glands. To evaluate the effect of lactogenic hormones on transgene expression, experiments with the primary culture of transgenic mammary explants were performed. It was revealed that the expression of transgenes was slightly increased by insulin plus dexamethasone or insulin plus prolactin treatment. However it was not increased by insulin, dexamethasone or prolactin (IDP) treatment alone. In contrast, the endogenous WAP gene was expressed only in the IDP treated group. These results demonstrate that MAR sequences are effective in improving the expression frequency of transgenes in transgenic mice although the developmental and hormonal regulations are not the same as those of the endogenous WAP gene.

Gene expression during involution of mammary gland (review).

After cessation of lactation, the mammary gland undergoes involution, regressing to a state resembling that of a virgin animal. This phase of mammary gland development is characterized by epithelial cell death and tissue remodeling. To understand molecular mechanisms of mammary gland involution, we identified involution-induced clones by differential screening of a mouse mammary gland cDNA library. Several known genes were induced during mammary gland involution: sulfated glycoprotein-2 (SGP-2), WDNM1, lactoferrin, ferritin heavy chain (FHC), lysozyme and osteopontin genes. Involution of the mammary gland is presumed to be mediated by a decrease in serum prolactin level induced by weaning, but may also involve changes in paracrine or autocrine growth factors. Effects of lactogenic hormones and EGF on the expression of the involution-induced genes were examined in mammary epithelial cells. Insulin, dexamethasone, and prolactin did not influence the expression of the FHC, WDNM1 and SGP-2 genes. However, EGF strongly inhibited the expression of WDNM1 and SGP-2 genes. Our recent results are reviewed and discussed.

Dietary energy restriction-mediated growth and mammary development in rats.

This research examined the extent to which dietary energy restriction modulates growth and mammary tissue composition during different developmental stages. Female rats were assigned to the following three dietary treatments: 1) ad libitum access to feed (AL), 2) 30% continuous energy restriction (CER), and 3) stair-step energy restriction (SSER). The SSER treatment consisted of an 8-wk, alternating schedule beginning with 60% energy restriction for 2 wk, followed by realimentation to feed offered for ad libitum intake for 2 wk. All treatments were initiated when rats were 5 wk of age. After the stair-step regimen, SSER rats were maintained on a 30% energy-restricted diet for the duration of the experimental period (25 wk of age). Rats reared on the energy restriction regimens weighed less and consumed less (P < .05) feed than controls, but they had feed efficiencies similar to those of controls. Energy restriction delayed the onset of puberty and retarded the growth of the offspring but had no effect on litter size. The overall values (averaged pregnancy through involution stages) of DNA, RNA, and RNA: DNA ratio (based on fat-free DM) and protein concentrations were similar in the mammary tissues of the energy restriction groups and those of the AL group. Lipid content in mammary tissue was generally decreased in the CER and SSER groups compared with the AL group. In summary, energy restriction delayed the onset of puberty and retarded the growth of the dam and progeny, but it did not affect mammary cellularity as it reduced fat deposition in the mammary gland.

Postmortem muscle glycolysis and meat quality characteristics of intact male Korean native (Hanwoo) cattle.

Five intact Korean bulls weighing about 550 kg were slaughtered to investigate postmortem glycolysis. Histochemical and meat quality characteristics of longissimus dorsi (LD) and psoas major (PM) muscles were made. Postmortem changes in ATP, glucose-6-phosphate and pH demonstrated that the rate of postmortem glycolysis in the PM was significantly faster than in the LD. The shear force to cut cooked PM was significantly lower than that of LD in 1 and 3 day aged samples, but no difference was observed between the two muscles in 7, 15 and 21 day aged samples. During the 21 days of aging, the rate of lipid oxidation, as measured by TBA value, was significantly faster in the PM than in the LD. The result suggests that PM muscle needs less aging time than LD muscle for optimum meat quality.

Role of compensatory growth in lactation: a stair-step nutrient regimen modulates differentiation and lactation of bovine mammary gland.

Twenty Holstein heifers were assigned to either a control or test (stair-step compensatory; food restriction, followed by refeeding) growth group. The stair-step growth model was designed to induce distinctive compensatory allometric mammary development during three different hormonal states, coinciding with prepubertal, pubertal, and late gestational stages. Mammary tissues obtained by biopsy from pregnant and lactating cows were used for acini culture and chemical composition analysis. Test mammary tissues from late gestation heifers contained less (P = .067) fat than control counterparts (731 vs. 628 mg/g). DNA, RNA, and protein contents in test mammary tissue from late gestation cows were higher (P = .001 to .088) compared to control tissue. Milk protein secretion of test acini in culture was increased more than 20% over that of the control acini. Lactating mammary acinar cells in culture from test cows exhibited a 14% increase in amino acid uptake over that of the control. RNA dot-blot hybridization analysis revealed that alpha s1- and beta-casein mRNA accumulation in acini from test tissue was increased (P = .027 to .042) as much as 40-50%. During both food restriction and refeeding, concentrations of plasma growth hormone were elevated. Food restriction decreased levels of plasma insulin, whereas levels of insulin were elevated during refeeding. The long-term influence of compensatory growth upon subsequent lactation performance was also evaluated. Milk production data derived from previous two sequential growth trials showed that cows from test groups produced approximately 10% more (P = .001) milk compared to control counterparts. Results reinforce our postulation that compensatory growth induced by nutritional modulation regulates the differentiation and functional activity of the mammary gland.

The effect of lactogenic hormones on protein synthesis and amino acid uptake in rat mammary acinar cell culture at various physiological stages.

1. The activities of protein synthesis and amino acid uptake at various physiological stages were determined by the incorporation of radioactive materials ([3H]-lysine, [14C]-cycloleucine) in rat mammary epithelial cell cultures. The activity of protein synthesis and amino acid uptake was higher in early lactation than in virgin, pregnant and late lactation stages. 2. Lactogenic hormones (prolactin, hydrocortisone and insulin) treatment related with mammary growth and differentiation increased the activities of protein synthesis and amino acid uptake. But increase of these activities was different at each physiological stage. 3. The effect of prolactin and hydrocortisone on the activities were greater in virgin, pregnant and late lactation than in early lactation. And effect of insulin was greater in pregnant and early lactation than in virgin and weanling.

Modulation of expression of fos and Ha-ras oncogenes and ornithine decarboxylase activity in mammary gland and liver of young female rats by the absence of dietary lipotropes.

There is evidence that diets deficient in lipotropes [methionine, choline, pteroylmonoglutamic acid (folic acid), and cyanocobalamin (vitamin B12)] induce and enhance hepatocarcinogenesis. This research examined the extent to which dietary lipotropes modify cellular oncogene expression and ornithine decarboxylase activity in mammary gland and liver of rats. Eighteen female Sprague-Dawley rats (8 wk old) were fed 3 wk on one of three diets: 1) a control synthetic diet; 2) a methyl-deficient diet lacking choline, methionine, pteroylmonoglutamic acid, and cyanocobalamin; or 3) a diet supplemented with twice the amount of each lipotrope as in the control synthetic diet. The group fed the methyl-deficient diet gained less body weight than groups fed the control or methyl-supplemented diet. The group fed the methyl-deficient diet had approximately 5- and 11-fold greater fos transcription in mammary gland and liver, respectively, than did the control group. The expression of the Ha-ras gene in mammary gland and liver of the group fed the methyl-deficient diet was increased by 4- and 6-fold compared with that of the control. Ornithine decarboxylase activity, considered to be a developmental marker, was higher in liver and mammary gland of the group fed the methyl-deficient diet than in either the group fed control synthetic diet or the group fed the methyl-supplemented diet. The methyl-deficient diet may have caused activation of the transcription factor fos and thus the activation of the transcription regulatory complex, AP-1. In turn, AP-1 may regulate genes, such as ornithine decarboxylase, which are responsible for cell proliferation and differentiation.

Lipotrope-modified diets enhance nitrosomethylurea-induced mammary carcinogenesis in female rats.

This study was conducted to determine the effects of lipotrope-modified (deficient or supplemented) diets on nitrosomethylurea- (NMU) induced mammary tumorigenesis. Eighty female Sprague-Dawley rats (4 wks old) were assigned to one of the following groups: control-synthetic diet (CSD), containing all required lipotropes; choline-methionine-deficient diet (CMD); methyl-deficient diet (MDD), lacking all lipotropes; and methyl-supplemented diet (MSD), containing twice as much of each lipotrope as the CSD diet. All animals were injected with NMU after a three-week dietary treatment period. MDD and MSD groups had shorter tumor latency periods (73 and 74 days, respectively) than the CSD group (105 days). Number of tumors per rat was significantly increased in the MDD group (4.6) compared with CSD (1.6), CMD (2.1), and MSD (2.5) groups. The results indicate that dietary manipulation of lipotropes in young female rats enhanced NMU-induced mammary tumorigenesis.