Metabolism of short-chain ceramide and dihydroceramide analogues in Chinese hamster ovary (CHO) cells

A series of radiolabelled ceramides (D-erythro and L-threo) and dihydroceramides (DL-erythro and DL-threo) with 2, 4 or 6 carbon N-acyl groups were synthesized. These analogues were incubated with cultured CHO cells and radioactive products isolated and analyzed. In addition to synthesis of short-chain sphingomyelin and glucosylceramide, radiolabelled sphingosine and sphinganine were released from short-chain ceramides and dihydroceramides and subsequently utilized for synthesis of long-chain ceramide and sphingolipids. Substrate preference for short-chain sphingomyelin synthesis in cells was D-erythro-ceramides > L-threo-ceramides > DL-erythro-dihydroceramides > DL-threo-dihydroceramides, and C4- and C6-analogues were preferred over the C2-analogue. Kinetic constants for conversion of short-chain (dihydro)ceramides to short-chain sphingomyelin were determined using CHO cell membranes and found to correlate with substrate preference in cultured cells. D-erythro-C6-Ceramide was the preferred substrate for short-chain glucosylceramide synthesis. D-erythro-C2-ceramide inhibited incorporation of [3H]serine into sphingomyelin, glucosylceramide and ceramide rapidly (2 h) and in a dose-dependent manner. Over a similar time period, [3H]choline-labelling of sphingomyelin was not affected. Inhibition of [3H]serine-labelling of sphingolipids appeared to correlate with release of [3H]long-chain bases from short-chain ceramides and dihydroceramides and synthesis of long-chain sphingolipids. However, some discrepancies between DL-erythro-C4- and C6-dihydroceramides, and D-erythro-C2-ceramide suggested that short-chain dihydroceramides were less efficient in suppressing de novo synthesis from [3H]serine, while contributing substantially to endogenous sphingolipid synthesis. Inhibition of de novo sphingolipid synthesis by short-chain ceramides and dihydroceramides could not be related to inhibition of serine palmitoyltransferase activity in vitro.     

Substrate specificity of prostate-specific antigen (PSA)

BACKGROUND: The serine protease prostate-specific antigen (PSA) is a useful clinical marker for prostatic malignancy. PSA is a member of the kallikrein subgroup of the (chymo)trypsin serine protease family, but differs from the prototypical member of this subgroup, tissue kallikrein, in possessing a specificity more similar to that of chymotrypsin than trypsin. We report the use of two strategies, substrate phage display and iterative optimization of natural cleavage sites, to identify labile sequences for PSA cleavage. RESULTS: Iterative optimization and substrate phage display converged on the amino-acid sequence SS(Y/F)Y decreases S(G/S) as preferred subsite occupancy for PSA. These sequences were cleaved by PSA with catalytic efficiencies as high as 2200-3100 M-1 s-1, compared with values of 2-46 M-1 s-1 for peptides containing likely physiological target sequences of PSA from the protein semenogelin. Substrate residues that bind to secondary (non-S1) subsites have a critical role in defining labile substrates and can even cause otherwise disfavored amino acids to bind in the primary specificity (S1) pocket. CONCLUSION: The importance of secondary subsites in defining both the specificity and efficiency of cleavage suggests that substrate recognition by PSA is mediated by an extended binding site. Elucidation of preferred subsite occupancy allowed refinement of the structural model of PSA and should facilitate the development of more sensitive activity-based assays and the design of potent inhibitors.     

Isolation and some properties of factor VIII (antihemophilic factor)

A method for isolation of factor VIII from cryoprecipitate fraction of human plasma has been elaborated. The isolation procedure involves precipitation with dextran, removal of fibrinogen by means of defibrase, precipitation with ammonium sulfate, polyethylene glycol fractionation, and Sepharose 6B gel filtration step. Factor VIII has been purified 7000- to 13,000 -- fold.. The preparation is homogenous by ultracentrifugal examination and it has an S20,w value of 19.4. It also shows a single precipitin line when subjected to immunoelectrophoresis employing rabbit antibodies against factor VIII. The preparation did not enter a 7.5% polyacrylamide gel containing sodium dodecyl sulfate even in the presence of 8 M urea. After reduction of the protein with 2-mercaptoethanol, subunits were formed which migrated as one band in polyacrylamide gel electrophoresis.     

Effect of the inducible nitric oxide synthase inhibitors aminoguanidine and L-N6-(1-iminoethyl)lysine on zymosan-induced plasma extravasation in rat skin

The effect of nitric oxide synthase (NOS) inhibitors on plasma extravasation in a rat model of zymosan-induced inflammation has been investigated. Plasma extravasation was determined in response to intradermal test agents over 0 to 45 min or 0 to 4 h by the accumulation of i.v. injected 125I-labeled human serum albumin. Zymosan (1-100 microg/site) produced a dose- and time-dependent plasma extravasation. N(G)-nitro-L-arginine methyl ester (30-300 nmol/site), but not aminoguanidine (AG; 10-300 nmol/site) or L-N6-(1-iminoethyl)lysine (L-NIL; 10-300 nmol/site), significantly (p < 0.01) inhibited zymosan-induced (10 microg/site) plasma extravasation over 0 to 45 min. However, both AG and L-NIL produced significant (p < 0.05) inhibition over 0 to 4 h. The inhibition produced by AG was reversed by i.v. L-arginine or by coinjection of the vasodilator, calcitonin gene-related peptide. Zymosan (10-100 microg/site) induced an increase in dermal blood flow (laser-Doppler flowmetry) and this was inhibited by AG. Neutrophils were depleted selectively with antiserum, but this did not affect plasma extravasation except at the highest dose of zymosan (100 microg/site). Furthermore, zymosan-induced edema was not modified at either time point by pretreatment with the cyclooxygenase inhibitor indomethacin (30 micromol/kg, s.c., -30 min). In conclusion, in this model of dermal inflammation, it is suggested that inducible NOS inhibitors selectively remove an inducible NOS component that, at least in part, acts to increase microvascular blood flow and thus the edema formation observed during 0 to 4 h. There is no evidence of a contributory role for neutrophils or cyclooxygenase products in this model.     

Substrate specificity in plasmalogen biosynthesis. Desaturation of 1-O-hexadecyloxyethyl-2-acylglycero-3-phosphoethanolamine in developing rat brain

1-O-[1'-14C]Hexadecyloxyethyl rac-glycerol was administered to 18-day-old rats by intracerebral injection, and incorporation of radioactivity into the brain lipids was determined after 6, 24 and 48 h. Some of the substrate was catabolized by oxidative cleavage of either of the two ether bonds. Cleavage in the hexadecyloxyethyl moiety yielded labeled palmitic acid, whereas oxidative cleaveage of the glycol glycerol ether bond produced O-hexadecyl glycolic acid. The substrate was also incorporated as such into both ethanolamine and choline phospholipids. Evidence is presented for the desaturation by rat brain of 1-O-hexadecyloxyethyl-2-acyl-sn-glycero-3-phosphoethanolamine to the plasmalogen analogue, while the corresponding choline phospholipid was not desaturated.     

Purification and some properties of ATP-citrate lyase from rat brain

ATP-citrate lyase has been purified from rat brain by a new procedure which yields an enzyme of specific activity of 21 U/mg protein (37 degrees C) (2050-fold purification). Purity (by sodium dodecyl sulfate-gel electrophoresis) of the preparation was comparable to that of rat liver ATP-citrate lyase of similar specific activity. Both brain and liver ATP-citrate lyase have the same electrophoretic mobility, as well as the same immunoreactivity against specific rabbit anti-rat liver ATP-citrate lyase antibody. These data indicate that rat brain ATP-citrate lyase is similar or identical to that present in rat liver. Intraperitoneally injected 32Pi was incorporated into the structural phosphate of ATP-citrate lyase in rat liver but not into the rat brain enzyme.     

Substrate specificity of 2-deoxy-scyllo-inosose synthase, the starter enzyme for 2-deoxystreptamine biosynthesis, toward deoxyglucose-6-phosphates and proposed mechanism

A crucial enzyme in the biosynthesis of the 2-deoxystreptamine aglycon of clinically important aminocyclitol antibiotics is 2-deoxy-scyllo-inosose synthase (DOIS), which is responsible for the initial carbocycle formation of 2-deoxy-scyllo-inosose (1) from D-glucose-6-phosphate (G-6-P) (2). To get more insight into the mechanism and substrate specificity, deoxy-D-glucose-6-phosphates (deoxy-G-6-P) were chemically synthesized and subjected to the reaction with DOIS. The enzyme appeared to use 2-deoxy- and 3-deoxy-G-6-P as substrates, both of which were converted into the corresponding dideoxy-scyllo-inosose products, but 4-deoxy-G-6-P failed in cyclization by DOIS. These results clearly support the proposed reaction mechanism involving the initial oxidation at C-4 of the G-6-P substrate. Another implication is the potential use of DOIS for the preparation of useful dideoxyinososes.     

Electrogenic properties and substrate specificity of the polyspecific rat cation transporter rOCT1

The previously cloned rat cation transporter rOCT1 detected in renal proximal tubules and hepatocytes (Gründemann, D., Gorboulev, V., Gambaryan, S., Veyhl, M., and Koepsell, H. (1994) Nature 372, 549-552) was expressed in Xenopus oocytes, and transport properties were analyzed using tracer uptake studies and electrophysiological measurements. rOCT1 induced highly active transport of a variety of cations, including the classical substrates for cation transport, such as N-1-methylnicotinamide, 1-methyl-4-phenylpyridinium (MPP), and tetraethylammonium (TEA), but also the physiologically important choline. In oocytes rOCT1 also mediated efflux of MPP, which could be trans-stimulated by MPP and TEA. Cation transport via rOCT1 was electrogenic. In voltage-clamped oocytes, transport of TEA and choline via rOCT1 produced inwardly directed currents, which were independent of extracellular ion composition or pH. The choline- and TEA-induced currents were voltage-dependent at nonsaturating concentrations, and the apparent affinity of these cations was decreased at depolarized voltages. Other substrates transported by rOCT1 were the polyamines spermine and spermidine. Interestingly, the previously described potent inhibitors of rOCT1, cyanine 863, quinine, and D-tubocurarine were substrates themselves. The data indicate that rOCT1 is an effective transport system that is responsible for electrogenic uptake of a wide variety of organic cations into epithelial cells of renal proximal tubules and hepatocytes.     

Divergence of beta-myosin heavy chain (betaMHC) expression in fetal rat cardiomyocytes in vitro and adult rat heart in vivo

The myosin heavy chain gene products are an important determinant of myocardial functional properties. Although a strong positive element (beta f1) within the betaMHC promoter region has previously been identified, to date no species comparisons in promoter strength have been made. To examine this question, we have used betaMHC deletion constructs, containing the rat or human beta f1 enhancer region, to determine expression both in vitro using rat fetal cardiomyocytes and in vivo by direct injection into adult rat heart. When reporter constructs were transfected into cultured fetal rat cardiomyocytes, the human beta reporter was expressed more than 3 fold above the equivalent rat construct. Exchange of the beta f1 enhancer indicated that the human sequence of the beta f1 enhancer was largely responsible. However, these findings were not replicated when the reporters were injected into the adult rat heart. In the adult myocardium the levels of reporter expression were similar for the betaMHC promoter reporters studied. These findings demonstrate a divergence between primary cardiomyocytes maintained in culture and the cardiomyocytes found within the intact adult heart.     

Classical conditioning in the fetal rat: Reinforcing properties of dynorphin A (1–13).

This study examined the reinforcing properties of dynorphin A (1-13) in a single-trial classical conditioning paradigm in the E20 rat fetus. Injection of dynorphin into the cisterna magna increased fetal motor activity and reduced facial wiping in a test of perioral cutaneous responsiveness. Dynorphin was effective as an unconditioned stimulus (US) in a classical conditioning paradigm using an artificial nipple conditioned stimulus (CS) and dynorphin A (1-13) US. The association between CS and US was dependent on activity in the kappa opioid system. Re-exposure to the artificial nipple CS after a single pairing of the nipple with dynorphin resulted in conditioned activation of the kappa opioid system. Dynorphin A (1-13) functions as a reinforcer for classical conditioning in the rat fetus after intracisternal or intrahemispheric injection with the conditioned response depending on route of administration and site of injection. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Ligands and activators of nuclear hormone receptors regulate epidermal differentiation during fetal rat skin development

Because a protective barrier is essential for life, the development of the epidermis and stratum corneum must be completed prior to birth. The epidermal permeability barrier is comprised of corneocytes embedded in a lipid enriched matrix. Recent studies from our laboratory, using an explant model of fetal rat skin development that closely parallels in utero development, have shown that hormones and other activators of members of the nuclear receptor family regulate permeability barrier ontogenesis by stimulating lipid metabolism and the formation of the extracellular lipid lamellae. Using this model we sought to determine whether these hormones and nuclear activators also regulate keratinocyte differentiation during fetal development. Profilaggrin/filaggrin and loricrin expression, assessed by in situ hybridization and by immunohistochemistry, were progressively increased during epidermal ontogenesis. Whereas profilaggrin/filaggrin and loricrin were not expressed at day 17 of gestation, by day 19 both were present in the upper layers of the epidermis and both became still more abundant by day 21. These developmental changes also occurred in fetal skin explants cultured in vitro for 4 d, although the expression levels did not appear as robust as in utero. Whereas neither profilaggrin/filaggrin nor loricrin were expressed in control explants cultured for 2 d, they were seen in explants treated with either thyroid hormone, glucocorticoids, or estrogens. In contrast, dihydrotestosterone treatment delayed the expression of profilaggrin/filaggrin and loricrin. Moreover, both clofibrate, a peroxisome proliferator-activated receptor-alpha ligand, and juvenile hormone III, a farnesoid X-activated receptor activator, markedly accelerated fetal epidermal differentiation, stimulating both profilaggrin/filaggrin and loricrin expression. Our results demonstrate that several hormones and activators of nuclear hormone receptors regulate epidermal differentiation during fetal development, affecting key constituents of both keratohyalin granules and the cornified envelope. Thus, a variety of ligands/activators of nuclear receptors accelerate not only permeability barrier ontogenesis, but also the expression of structural proteins essential for stratum corneum formation.     

Human (and some other primates') uterine teres ligament represents a mammalian developmental novelty

BACKGROUND: The primordia of the structures developing into the mammalian male cremaster sacs emerge as well in females fetuses. In most species the structures developing from these primordia in females remain inconspicuous: the so-called uterine teres ligament consisting of a slender part across the uterine broad ligament and a more or less developed bulbous structure at the site where this ligament inserts into the inguinal abdominal bottom. Not many data are available concerning the growth, development, or function of the uterine teres ligament. In humans--and also in other "higher" female primates--the uterine teres ligament is a major structure consisting mainly of smooth musculature. It is attached to the ventral aspect of the tubo-uterine junction. From there it courses across the uterine broad ligament and extends, retroperitoneally, to the inguinal region where it pierces through the inguinal canal to end in the tissues ventral of the pubic bones. OBSERVATIONS: Analysis of the fetal development of the human uterine teres ligament, as compared with that of various other non-primate mammals, offers an explanation for its unusual anatomical condition. Evidence is conferred that, in human fetuses, there is no counterpart for the slender ligament across the broad ligament in other mammals. Instead, the homologue of the rudimentary bulbous structure in the abdominal bottom of non-primate females develops into a strong muscular structure which is directly connected to the (para-)menonephric duct wall. CONCLUSION: It is concluded that the human uterine teres ligament is to be judged a structure different from that of other, non-primate, mammals. It is speculated that the unusual structure of the human teres ligament is related to one or more of the many unusual features of human uterine development: as a single organ (uterus simplex), with a position deep in the abdominal cavity below the pelvic brim, and far away from the posterior abdominal wall. The unusual anatomical position may require an unusual construction of the uterine suspensory apparatus of which the teres ligament is one component.     

A multiple-level, comprehensive approach to the prevention of fetal alcohol syndrome (FAS) and other alcohol-related birth defects (ARBD)

A comprehensive program for the prevention of fetal alcohol syndrome (FAS) and alcohol-related birth defects (ARBD) must consider multiple approaches and utilize knowledge from a variety of academic disciplines. Issues related to culture, society, behavior, belief systems, and medicine must all be considered for both etiology and solutions. A broad paradigm such as a public health model integrates various elements of approach. Because FAS and other levels of ARBD form a spectrum, from severe to negligible damage, a variety of drinking patterns with various characteristics and etiologies have to be addressed. This paper describes a multiple-level, comprehensive program with primary, secondary, and tertiary prevention components. Practical recommendations are proposed for addressing ARBD in a variety of arenas. While secondary and tertiary prevention hold promise for short-term reduction of FAS and ARBD prevalence, comprehensive prevention serves both short- and long-term effects. Multiple level prevention efforts are well served by clear and compelling vision and mission statements, and require careful evaluation.     

Immunohistochemical characterization of transplantable rat squamous cell carcinoma (FF-6) in skin and thymus

FF-6 is a transplantable squamous cell carcinoma which originally arose in the facial skin of a DA rat. It was established after maintaining the tumor in the subcutaneous tissue or peritoneal cavity of DA rats conventionally for over 30 generations. When the soybean-sized original FF-6 tumor was transplanted subcutaneously, it became an oval, hard, whitish, solitary and thumb-head-sized nodule within one month. After intraperitoneal transplantation of FF-6, it formed many nodules ranging from miliary to thumb-head size, which adhered and/or metastasized to many abdominal organs. When FF-6, cut into small pieces, was injected into the lower lip, the tumor grew bigger in situ, and metastasized to regional lymph nodes. Histologically, FF-6 was characterized as a well-differentiated squamous cell carcinoma, showing positive staining with anti-keratin, anti-laminin, anti-collagen type IV, anti-fibronectin and UB-14 antibodies. This transplantable tumor may be useful for analyzing the mechanisms of proliferation and metastasis of squamous cell carcinoma in vivo, and the host defence mechanism in rats, as well as being a suitable model of human squamous cell carcinoma.