Search

A hybrid mouse major urinary protein (MUP)/SV40 T antigen gene was microinjected into fertilized mouse embryos and the resulting transgenic mice analyzed for the regulated expression of the transgene. Available evidence indicates that the MUP gene used for the hybrid gene construct is expressed in both male and female liver and possibly mammary gland. Three different transgenic lines exhibited a consistent pattern of tissue specific expression of the transgene. As a consequence of transgene expression and T antigen synthesis in the liver, both male and female transgenic animals developed liver hyperplasia and tumors. Transgene expression and liver hyperplasia commenced at approximately 2‐4 weeks of age, the same time that MUP gene expression is first detected in the liver. The expression of the transgene resulted in an immediate strong suppression of liver MUP mRNA levels but had relatively little effect on other liver specific mRNAs. From 4 to 8 weeks, the liver increased several fold in size, relative to non‐transgenic littermates. Definitive tumor nodules were not apparent until 8‐10 weeks. The transgene was also consistently found to be expressed in the skin sebaceous glands and the preputial gland, a modified sebaceous gland. The expression of the transgene in the skin sebaceous glands is consistent with the presence of MUP mRNA in the skin and a putative role for MUPs in the transport and excretion of small molecules. Occasional expression of the transgene in other tissues (kidney and mammary connective tissues) was also noted.(ABSTRACT TRUNCATED AT 250 WORDS)

Galactosyltransferase (EC 2.4.1.22) requires bivalent metal ions for its activity. However, preparations of this enzyme solubilized from Golgi membranes of lactating rat mammary gland were shown to be activated not only by Mn2+, Ca2+ and Mg2+, but also by spermine, spermidine, lysyl-lysine, ethylenediamine and other diaminoalkanes, and by a range of basic proteins and peptides, including clupeine, histone, polylysine, ribonuclease, pancreatic trypsin inhibitor, cytochrome c, melittin, avidin and myelin basic protein. Both N-acetyl-lactosamine synthetase and lactose synthetase activities were enhanced. A basic protein fraction was isolated from bovine milk and shown to activate galactosyltransferase at low concentrations. The polyanions ATP, casein, chondroitin sulphate and heparin reversed the activation of galactosyltransferase by several of the above substances. Galactosyltransferase, assayed as a lactose synthetase, showed a 10-fold greater affinity for glucose when Mn2+ ions were replaced by clupeine or by ribonuclease as cationic activator. Evidence was obtained for the presence of an endogenous cationic activator in solubilized Golgi membrane preparations which evoked a similar low apparent Km, glucose. The findings are discussed in the light of cationic activations of glycosyltransferases generally, of the porous nature of the Golgi membrane, and of the unlikelihood of bivalent metal ions being the physiological activators of galactosyltransferase. It is suggested that the natural cationic activator of lactose synthetase may be a secretory protein acting in a manner analogous to the enzyme's activation by alpha-lactalbumin. A scheme is proposed for the two-stage synthesis of lactose and phosphorylation of casein within the cell, to accommodate the apparent incompatibility of these two processes.

Arteriovenous glucose difference across the mammary gland of the lactating rat was used as an ‘instantaneous’ monitor of mammary glucose uptake. Plasma [glucose] and arteriovenous glucose difference varied according to whether Halothane, diethyl ether or sodium pentobarbitone anaesthesia was used. In pentobarbitone-treated rats a 60% glucose extraction in the fed state decreased to 5% after 18 h starvation, and recovered to 40% and 59% after 15 min and 60 min re-feeding respectively. The increase and decrease in plasma [fatty acids] and the depletion and restoration of hepatic glycogen mostly followed similar time courses. Re-feeding was accompanied by a brief surge of plasma [insulin]. Starved lactating rats showed a markedly greater capacity than age-matched virgin rats in the oral and intraperitoneal glucose tolerance tests. Mammary glucose uptake in the starved rat was significantly restored by oral or intraperitoneal glucose or by insulin, but not by acetoacetate or by heparin-induced elevation of plasma [fatty acids]. The role of insulin and of possible changes in mammary sensitivity to insulin in the return of mammary glucose uptake on re-feeding is discussed.

The uptake and release of 3-O-methyl-D-[3H]glucose at 37 degrees C by acini, prepared from lactating-rat mammary gland with collagenase, was inhibited by glucose, phloretin, cytochalasin B, HgCl2 and low temperature. Uptake and phosphorylation of 2-deoxy-D-[3H]glucose, studied in greater detail, could be ascribed to a specific, saturable, inhibitable, process of apparent Km 16 mM and Vmax. approx. 56 nmol/min per mg of protein, plus a non-specific, non-inhibitable process that was monitored with [14C]fructose. The mean rate of uptake of 5 mM-2-deoxyglucose (16 nmol/min per mg of protein) was similar to the rate of consumption of 5 mM-glucose, suggesting that transport was a rate-limiting step in the overall metabolism of glucose. This accords with evidence for a glucose gradient across the plasma membrane.

1. Purified Golgi-membrane vesicles of lactating-rat mammary gland were penetrated by glucose. 3-O-methylglucose, mannose, fructose, sorbitol and mannitol, but not by lactose or sucrose. 2. The kinetics of mannitol uptake and release were followed at 2-6 degrees C with the aid of fine filters (0.45 micrometers pore size) to separate the vesicles from the medium. 3. Mannitol efflux exhibited apparent first-order kinetics with k approximately 1 min-1. Neither saturability, nor inhibition by excess sorbitol or glucose, could be observed. 4. Mannitol efflux at 18 degrees C was about seven times faster than at 1 degrees C, and rates at higher temperatures were too fast to be measured. The rate of glucose efflux at 2-6 degrees C exceeded that of mannitol severalfold. 5. These findings imply a channel or carrier of definite, but limited, specificity straddling the Golgi membrane and able to supply glucose for lactose synthesis.

1. The ability of phlorrhizin to inhibit the galactosylation of glucose was re-examined with Golgi membrane vesicles purified from rat mammary gland, and extended to the galactosylation of several glucose analogues and N-acylglucosamines. 2. The inhibition is ascribed, contrary to previous conclusions, to a general annealing of leaky membranes comprising a minority of the vesicles. 3. Three thiol reagents were able to inhibit the galactosylation of N-acylglucosamines with less, or no, inhibition of galactosylation of glucose. This demonstrates the existence of a Golgi membrane carrier that distinguishes between glucose and N-acylglucosamines.

1. UDP-galactose utilization by isolated Golgi vesicles or rat mammary gland synthesizing lactose causes accumulation of UMP but not UDP, although UDP is the immediate product of lactose synthase (EC 2.4.1.22). 2. This can be ascribed to a nucleoside diphosphatase (EC 3.6.1.6), specific for UDP, GDP and IDP, activated by bivalent metal ions and apparently located on the luminal face of the Golgi membrane. 3. The uridine diphosphatase activity exceeds the total galactosyltransferase activity 5-fold, and is estimated to maintain UDP at about 14 micrometer within the Golgi lumen. 4. Evidence is given that UMP, but not UDP, penetrates the membrane and that UMP is rephosphorylated to UDP by a UMP kinase located in the cytosol. 5. Golgi-cytosol relationships with respect to lactose synthesis are formulated in terms of a uridine nucleotide cycle which throws new light on the energy cost and possible regulation of lactose synthesis.

1. The previously reported induction of luteal 20α-hydroxy steroid dehydrogenase by administration of aminoglutethimide to late-pregnant rats was shown to be unaffected by prior removal of the foetuses. Aminoglutethimide therefore does not act via the foetuses in this context. 2. The ability of injected oestrogen to prevent the above induction was lost by delaying the injection for 12h after aminoglutethimide, although the increase in enzyme activity begins only after 24h. 3. Induction of 20α-hydroxy steroid dehydrogenase by foetoplacental removal on day 18 of pregnancy was inhibited by human choriogonadotropin, lutropin (luteinizing hormone) and pregnant-mare serum gonadotropin, but not by somatotropin (growth hormone), thyrotropin or follitropin (follicle-stimulating hormone) 4. Indomethacin blocked the normal induction of 20α-hydroxy steroid dehydrogenase in late pregnancy and that caused by aminoglutethimide. It partially blocked that caused by human choriogonadotropin given on days 19-20 and that caused by 2-bromo-α-ergocryptine on days 5-6, but failed to block that caused by human choriogonadotropin on days 15-16 or by foetoplacental removal on day 18 of pregnancy. 5. These findings, and the control of progesterone synthesis in late pregnancy, are interpreted in terms of a sequence of hormonal or enzymic syntheses, each of which is inhibited by the product of the preceding synthesis.

1. At short incubation times, and under suitable osmotic conditions, the lactose synthesized by Golgi-derived vesicles of rat mammary gland is 85-90% particulate. Evidence is presented for its occlusion within the lumen of the vesicles. 2. Ovalbumin is used as a bulky active-site inhibitor to show that the active site of lactose synthase lies on the inner face of the Golgi membrane. 3. Phlorrhizin and phloretin inhibit lactose synthesis by such vesicles, indicating the presence of a glucose-transport system. 4. The relationship of this topography to the synthesis of N-acetylneuraminyl-lactose and to the secretion of milk sugars is discussed.

Let $ S(V)$-dimensional representation $ V$ $ {\text{G}}{{\text{L}}_n}{{\mathbf{F}}_q}$The multiplicity series in $ S(V)$ $ {\text{G}}{{\text{L}}_n}{{\mathbf{F}}_q}$ $ {F_{{\text{S}}\text{t}}}(t) = \sum\nolimits_{k = 0}^\infty {{a_k}{t^k}} $ is the multiplicity of St in the $ k$. We show that $ {F_{{\text{S}}t}}(t) = {t^r}\prod\nolimits_{i = 1}^n {{{(1 - {t^{{q^i} - 1}})}^{ - 1}}} $ $ r = \sum\nolimits_{i = 1}^{n - 1} {({q^i} - 1} )$ of the parabolic subgroups of .

Summary.The sustained elevation during Days 12–21 of pregnancy of rat luteal ornithine decarboxylase, which is inducible experimentally by hCG and PGF-2α, was dependent upon intact placentae and oestrogen, but independent of prostaglandin, and was absent at corresponding times of pseudopregnancy. It is suggested that this enzyme activity is a response to an hCG-like hormone of placental origin. Two brief reappearances of enzyme activity on Days 21 and 22 of pregnancy, dependent upon intact placentae and upon both oestrogen and prostaglandin, suggest a pulsatile release of PGF-2α from the uterus or placentae. These experiments illustrate the value of ornithine decarboxylase in seeking unknown hormonal influences upon the corpus luteum.

The ability of rat mammary-gland Golgi membranes to produce monosaccharide-specific pores in phospholipid vesicles was investigated. The apparent ability of Triton X-100 extracts of Golgi membranes to form such pores was re-evaluated, since we have now found that an apparent pore is produced by the detergent alone. We therefore incorporated intact Golgi membranes (1 mg of protein) into egg-yolk phospholipid vesicles by direct sonication in the absence of any detergent. These vesicles retained about 0.6% of the total sucrose, but demonstrated selective permeability towards glucose compared with sucrose, with 19.8% of the glucose being lost during gel filtration on Sepharose 4B. This phenomenon seemed to be enhanced by the presence of acidic phospholipids and lysophosphatidylcholine, but was inhibited by inclusion of cholesterol in the vesicles. The best mixture of phospholipids comprised 6.5 mg of egg-yolk phospholipid, 1 mg of phosphatidylserine and 0.05 mg of lysophosphatidylcholine, where 32.9% of the glucose was lost. By using this optimum phospholipid mixture the pores were shown to be permeable to both glucose and mannitol, whereas sucrose and lactose were retained by the vesicles. Chaps (3- [(3-cholamidopropyl)dimethylammonio] propane-1-sulphonate)-solubilized membranes produced similar permeability in vesicles produced by dialysis of a solution of the phospholipids mixed with the membrane extract. This technique resulted in a greater loss of glucose, 33% loss requiring about 0.25 mg of protein. The pore-forming ability of both intact Golgi membranes and Chaps extracts was sensitive to boiling and proteolysis, indicating that a membrane protein was likely to be involved in pore formation.

The fructose 2,6-bisphosphate (Fru-2,6-P2) content and intracellular concentration of lactating mammary gland was measured in fed, starved and re-fed rats. There was little or no change on starvation, and about 1.5-fold rise on re-feeding, contrasting with estimated glycolytic changes of about 10-fold. The 6-phosphofructokinase (PFK-1) activity of mammary extracts was highly sensitive to added Fru-2,6-P2 under all conditions examined, and appeared to approach saturation at physiological concentrations of this effector. The activity of mammary PFK-1 measured under optimal and ‘physiological’ conditions suggested that this enzyme operates in vivo at about 24% of maximal rate, and is likely to be an important rate-limiting factor in mammary glycolysis.

Lactose synthesis and fatty acid synthesis in intact lactating-rat mammary gland were measured simultaneously by incorporation of [U-14C]glucose and of both [U-14C]glucose and 3H2O respectively. Both processes were almost abolished by overnight starvation. Self-re-feeding caused recovery of lipogenesis to 100% of normal by 2 h and to 170% by 5 h. Lactose synthesis recovered to 80% of normal by 5 h. Food intubated to starved rats caused partial recovery in 3 h, standard diet favouring lactose synthesis and sugars favouring lipogenesis. Casein and starch were ineffective. Olive oil intubated to fed rats suppressed lipogenesis greatly and lactose synthesis slightly. Paraffin oil or water partly mimicked these effects. Adrenaline (subcutaneous) decreased lipogenesis from glucose, whereas insulin (subcutaneous) caused hypoglycaemia associated with loss of lactose synthesis but unchanged fatty acid synthesis. Streptozotocin and 2-bromo-alpha-ergocryptine (CB-154) impaired lipogenesis but not lactose synthesis. The results are interpreted in terms of competition for intracellular glucose by biosynthetic pathways for lactose and fat, and the possible implications for variations in milk composition are discussed.

The Michaelis-Menten equation for the utilization of competing substrates was applied to the uptake of 2-deoxy[3H]glucose into the mammary gland of anaesthetized lactating rats. Intracellular water was calculated from total tissue water and sucrose space. Fed rats had a mean transport capacity of 2.2 mumol/min per g of tissue, giving an actual glucose transport in vivo of 1.1 mumol/min per g. Transport decreased by 90% on overnight starvation and returned to normal by 2 h of re-feeding. Similar changes were observed in the 1 min or 5 min transport of circulating 3-O-methylglucose. Transport of 3-O-methylglucose in starved rats was restored towards normal by insulin. In fed rats it increased between parturition and day 12 of lactation. The findings support the proposal that transport is a rate-limiting factor in the mammary utilization of carbohydrate.

Golgi-membrane vesicle penetration was studied by osmotic lysis. Pronounced temperature dependence of mannitol and mannoheptitol penetration over 0-37 degrees C gave linear Arrhenius plots, with activation energies of 75 and 117 kJ/mol respectively. Glucose penetration was constant over pH 5-9, but was respectively faster and slower at higher and lower pH values. Solubilized, dialysed, heat-stable extracts of Golgi membrane were reconstituted into egg yolk phospholipid membranes with apparent recovery of specific permeability. Penetration is interpreted in terms of a pore, for which the Renkin equation predicts a radius of about 0.54 nm.

Mouse Ltk- cells were stably transfected with cloned genes encoding the mouse major urinary proteins (MUPs). C57BL/6J MUP genomic clones encoding MUP 2 (BL6-25 and BL6-51), MUP 3 (BL6-11 and BL6-3), and MUP 4 (BL6-42) have been identified. In C57BL/6J mice, MUP 2 and MUP 4 are known to be synthesized in male, but not female, liver, and MUP 3 is known to be synthesized in both male and female liver and mammary gland. A BALB/c genomic clone (BJ-31) was shown to encode a MUP that is slightly more basic than MUP 2 and was previously shown to be synthesized in both male and female liver of BALB/c but not C57BL/6 mice. Comigration on two-dimensional polyacrylamide gels of the MUPs encoded by the transfecting gene provides a basis for tentative identification of the tissue specificity and mode of regulation of each gene. DNA sequence analysis of the 5' flanking region indicates that the different MUP genes are highly homologous (0.20 to 2.40% divergence) within the 879 base pairs analyzed. The most prominent differences in sequence occur within an A-rich region just 5' of the TATA box. This region (from -47 to -93) contains primarily A or C(A)N nucleotides and varies from 15 to 46 nucleotides in length in the different clones.

Luteolysis was induced in rats during late pregnancy by fetoplacental removal, and was monitored by the increased activity of luteal 20α-hydroxysteroid dehydrogenase (20α-OHSDH). The extent of enzyme induction over a given length of time varied according to the time of day at which the fetuses and placentae were removed, 11.00 and 21.00 h appearing to give optimal and minimal enzyme activities respectively. The 20α-OHSDH was more readily induced on day 19 than on day 18.