Antagonism between Nur77 and glucocorticoid receptor for control of transcription.

Two important functions of glucocorticoids (Gc), namely, suppression of immune system function and feedback repression of the hypothalamo-pituitary-adrenal (HPA) axis, are mediated through repression of gene transcription. Previous studies have indicated that this repression is exerted in part through antagonism between the glucocorticoid receptors (GR) and the AP-1 family of transcription factors. However, this mechanism could not account for repression of the pro-opiomelanocortin (POMC) gene, an important regulator of the HPA axis. Our recent identification of the orphan nuclear receptor Nur77 as a mediator of CRH induction of POMC transcription led us, in the present work, to show that Gc antagonize this positive signal at two levels. First, Gc partly blunt the CRH induction of Nur77 mRNA, and second, they antagonize Nur77-dependent transcription. GR repression is exerted by antagonism of Nur77 action on the NurRE element of the POMC gene. Gc antagonism of NurRE activity was observed in response to physiological stimuli in both endocrine (CRH induction of POMC) and lymphoid (T-cell receptor activation) cells. In transfection experiments, transcriptional activation by Nur77 and the repressor activity of liganded GR titrated each other on their cognate DNA target. In vitro binding experiments as well as mutation analysis of GR suggest that the mechanism of GR antagonism of Nur77 is very similar to that of the antagonism between GR and AP-1. The convergence of positive signals mediated by Nur77 (and also probably by related family members) and negative signals exerted by GR appears to be a general mechanism for control of transcription, since it is active in both endocrine and lymphoid cells.

Direct visualization of lipid deposition and reverse lipid transport in a perfused artery : roles of VLDL and HDL.

The major goal of this study was to determine the interactions of VLDL surface and core lipids with the artery wall. We first demonstrated in vitro that surface lipid in VLDL could be traced using the phospholipid-like fluorescent probe 1,1'-dioctadecyl-3,3, 3',3'-tetramethyl-indocarbocyanine (DiI). The core of VLDL particles was traced by fluorescently labeling apolipoprotein B with TRITC. The labeled VLDLs were perfused through rat carotid arteries, and accumulation of the fluorescently labeled VLDL components in the arterial walls was determined by quantitative fluorescence microscopy. Addition of lipoprotein lipase increased the accumulation of both DiI and TRITC by >2.3-fold. Histological examination showed that DiI and TRITC were primarily localized to the endothelial layer; however, DiI also accumulated as small "lakes" deeper in the artery, in a subendothelial position. Addition of HDL to the perfusion decreased the accumulation of surface lipid and apolipoprotein B-containing particles and eliminated the DiI lakes. Moreover, the increase in endothelial layer permeability associated with lipolysis was attenuated 21% by HDL. If VLDL surface lipid first was allowed to accumulate in the arterial wall, its subsequent rate of loss was more than twice as fast if HDL was included in the perfusate. These studies directly demonstrate atherogenic effects of VLDL lipolysis and their inhibition by HDL.

Characterization of the estrogen receptor in two antiestrogen-resistant cell lines, LY2 and T47D.

Drug resistance occurs frequently during breast cancer treatment with antiestrogens. Since antiestrogen action is mediated by the estrogen receptor (ER), we have examined both the structural and functional properties of the ER present in two breast cancer cell lines, LY2 and T47D, which proliferate rapidly in the presence of antiestrogens. The ER function in LY2 cells was indistinguishable from that of the parental tamoxifen-sensitive MCF-7 cells as assessed by estrogen regulation of two endogenous genes and estrogen-regulated transcription in a transient transfection system. RNase protection assays, sensitive enough to detect single base pair mismatches, showed that the sequence of the coding region of ER of LY2 and T47D cells was wild type. Thus the ER appears to be normal in two independently isolated breast cancer cell lines whose growth is resistant to the inhibitory effect of antiestrogens. Moreover by conducting the cell proliferation studies in a phenol red-free medium, we have demonstrated that the antiestrogen resistance of LY2 and T47D cells corresponds in fact to an estrogen-independent growth.

Wild-type egr1/Krox24 promotes and dominant-negative mutants inhibit, pluripotent differentiation of p19 embryonal carcinoma cells.

The zinc-finger transcription factor Krox24 was analysed for its role in differentiation in P19 embryonal carcinoma cells. Reciprocal dominant negative mutants consisting of Krox24 deleted for a crucial region of the zinc-finger domain (delta Krox24) or of the zinc-finger region alone (delta Krox24Zf) abolished the activation of transcription by Krox24 in P19 cells. Expression of Krox24 led to spontaneous differentiation of P19 cells in a lineage-independent fashion. Krox24 transfected populations, as well as individual clones randomly picked from them, displayed a wide array of diverse morphologies and expressed markers characteristic of a variety of differentiated cells. The dominant negative mutants blocked differentiation of P19 cells. We conclude that expression of Krox24 is sufficient for pluripotent differentiation of embryonal carcinoma cells, and that expression of Krox24 or other egr family members is essential to this process.

Cooperativity in Bacillus stearothermophilus pyruvate kinase.

The enzyme pyruvate kinase (PK) from the moderate thermophile Bacillus stearothermophilus has been used as a model system with which to investigate the homotropic and heterotropic cooperative interactions of the enzyme. Cooperative ligand binding by the wild-type enzyme was measured using pre-steady-state and steady-state fluorescence spectroscopy, and steady-state kinetics. The results suggest that the cooperative structural changes induced by the substrate phosphoenolpyruvate (PEP) are distinct from those induced by the allosteric activator ribose- 5-phosphate (R5P). Furthermore the structural transition induced by the binding of saturating amounts of both PEP and R5P is itself distinct. This conclusion was further substantiated by the production of five mutant proteins in which the R5P- and PEP-induced homotropic cooperative transitions were separated. These results suggest that the cooperativity exhibited by pyruvate kinase from B. stearothermophilus does not conform to a simple two-state model. A putative four-state model is proposed.

Estrogen receptor synthesis and turnover in MCF-7 breast cancer cells measured by a density shift technique.

The level of estrogen receptor (ER) is a major factor regulating cell sensitivity to estrogen. We have determined the rates of ER synthesis and turnover in MCF-7 breast cancer cells by incubating cells in medium supplemented with 13C15N2H-amino acids (dense amino acids) and monitoring the shift from "old-light" (preexisting) to "new-dense" (newly synthesized) receptors by velocity sedimentation on 0.4-M KCl, 5-20% sucrose gradients prepared in buffered deuterium oxide. Cytosol or unoccupied nuclear ER prepared from control cells grown in the presence of 12C14N1H-amino acids (normal amino acids) and labeled in vitro with [125I]iodoestradiol sediments as a single, normal density peak. When cells were incubated in medium supplemented with dense amino acids for various periods of time, the [125I]iodoestradiol-labeled receptor showed a progressive shift with time to a denser species; by 3 h, a more rapidly sedimenting shoulder was observed on the normal density peak; by 6 h, 40% of the receptor sedimented at the normal density rate, with 60% at a more rapid rate; by 8 h, the predominant peak (70%) was at the more dense position, and at 15 h, all of the sites sedimented as the denser form. Thus, the magnitude of the receptor peak of normal density as a function of time in dense medium indicates that the receptor in control cells has a half-life of 4.0-4.5 h. To determine whether estradiol or the antiestrogens nafoxidine (U11,100A; (1-(2-[p-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy] ethyl)pyrrolidine hydrochloride) or CI628 affected the turnover rate of the receptor, measurements were performed on cells grown in the continuous presence of estradiol or antiestrogen (steady state conditions). Exposure of cells to 10 nM estradiol was found to increase the turnover rate of the nuclear receptor, while exposure to 200 nM nafoxidine or CI628 (alpha-(p-[2-(1-pyrrolidino)ethoxy]phenyl)4-methoxy-alpha'- nitrostillbene) did not substantially alter the turnover rate of the nuclear receptor. The half-lives of receptor were found to be 4.02 +/- 0.23 and 4.47 +/- 0.26 h for control unoccupied cytosol and nuclear receptors, 3.00 +/- 0.38 h for estradiol-occupied nuclear receptors, 4.90 +/- 0.66 h for CI628-occupied nuclear receptors, and 3.43 +/- 0.37 h for nafoxidine-occupied nuclear receptors. These results indicate that ER turns over rapidly, with a half-life of 3-5 h, in the presence or absence of estradiol or antiestrogen, and that receptor synthesis is also rapid, with the rate of appearance of newly synthesized receptor being 0.3-0.5 pmol/mg DNA . h. These rates provide the cell with the capacity for dynamic and rapid regulation of its ER levels.

Characterization of the subunit nature of nuclear estrogen receptors by chemical cross-linking and dense amino acid labeling.

We have used chemical cross-linking and dense amino acid labeling of estrogen receptors to characterize the subunit nature and rate of turnover of nuclear 5S estrogen-receptor complexes. When MCF-7 human breast cancer cells are incubated with [3H]estradiol or [3H]antiestrogen [alpha-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-alpha'-nitrostilbe ne (CI628M) or (Z)-1-[4-(2-[N-aziridinyl]ethoxy)phenyl] 1,2-diphenyl-1-butene (tamoxifen aziridine)] and nuclear estrogen-receptor complexes are extracted with 0.6 M KCl and then chemically cross-linked with the cross-linker 2-iminothiolane, the cross-linked receptor complexes sediment as a 5.4S species on 3 M urea-containing sucrose gradients, while the noncross-linked species are 4S. Sodium dodecyl sulfate-polyacrylamide gel analyses of these cross-linked nuclear receptor complexes labeled with the covalently attaching ligand [3H]tamoxifen aziridine reveal a species of about 130,000 mol wt, while the noncross-linked or the cross-linked but mercaptan-cleaved receptor is 65,000 mol wt. Both receptor species are also detectable by interaction with an immunoadsorbent column containing antireceptor monoclonal antibody. For analyses of receptor turnover rates, cells exposed for different time periods to medium containing dense (15N, 13C, and 2H) amino acids were labeled with [3H]antiestrogen [1-[4-(2-dimethylaminoethoxy)phenyl]1-[4-hydroxyphenyl] 2-phenylbut-1-(2)ene (trans-hydroxytamoxifen) or CI628M] or [3H]estradiol, and salt-extracted nuclear estrogen receptors were analyzed on sucrose gradients. The normal density 5S form shifted to a broader, more dense peak at 2 and 4 h and finally, by 8-10 h, to a more dense, sharply sedimenting species. The time course of this shift is the same as that seen for the 4S urea-dissociated nuclear receptor form (t1/2 approximately 4h), suggesting that the 5.4S nuclear receptor is composed of two species which turn over at the same rate. We conclude from these cross-linking and density shift experiments that the nuclear 5S receptor complex consists of two similarly sized units, which turn over with similar half-lives. These data provide strong evidence that the 5S nuclear receptor complex is a homodimer of two 4S, 65,000 mol wt monomers.

Time utilisation pattern of staff of two primary health centres in Ballavgarh, Haryana.

BACKGROUND: All National health programmes are implemented through the Primary Health Centre staff. Targets for the year 2000 A.D. have been fixed for different programmes. Some programmes are getting more emphasis, perhaps at the cost of others. The study area has already achieved most of the targets set for 2000 A.D. Studying the time utilisation pattern of the workers of these PHCs can give valuable information for planning of working of other PHCs. OBJECTIVE: To study the time utilisation pattern of the staff of the two PHCs run by Centre for Community Medicine, AIIMS. METHODS: The multipurpose workers (MPWs) and the health assistants (HAs) were accompanied by investigators and information collected regarding their utilisation of time in the field. The Medical Officers were asked to maintain a diary from which this information was collected RESULTS: The MPWs spend about 3.3 minutes in each house. Child care (immunisation, Vit. A and folifer distribution) is the main activity being carried out by both male as well as female worker. Other important activities for male worker are: family welfare (18%), malaria work (11%) and collection of vital statistics (10%). For the female worker Antenatal care (25%) and family welfare (20%) were other important activities. For the HAs also child care was an important activity. However for the male HA malaria related work was the most important. The Medical Officer spends about 60% of this time in administrative and supervisory work. CONCLUSIONS: Immunisation programme is getting the maximum input from workers, which is reflected in > 90% coverage of all vaccines. Family Welfare and Tuberculosis activity are not getting the emphasis which they deserve. Some rethinking about the strategy is essential if all round progress in achieving the targets for the year 2000 A.D. is to be made.

Genetic control of susceptibility to Candida albicans in susceptible A/J and resistant C57BL/6J mice.

The importance of host factors in determining susceptibility to systemic Candida albicans infections is evident in both humans and mice. We have used a mouse model to study the genetic basis of susceptibility, using the inbred strains A/J and C57BL/6J, which are susceptible and resistant, respectively, based on different parameters of the response to infection. To identify genes responsible for this differential host response, brain and kidney fungal load were measured in 128 [A/J x C57BL/6J] F(2) mice 48 h after infection with 5 x 10(4) C. albicans blastospores. Segregation analysis in this informative population identified complement component 5 (C5/Hc) as the major gene responsible for this differential susceptibility (LOD of 22.7 for kidney, 19.0 for brain), with a naturally occurring mutation that causes C5 deficiency leading to enhanced susceptibility. C5 was also found to control heart fungal load, survival time, and serum TNF-alpha levels during infection. Investigation of the response to C. albicans challenge in a series of AcB/BcA recombinant congenic strains validated the importance of C5 in determining the host response. However, the strains BcA67 and BcA72 showed discordant phenotypes with respect to their C5 status, suggesting additional complexity in the genetic control of the inter-strain difference in susceptibility observed in A/J and C57BL/6J following systemic infection with C. albicans.

High-resolution linkage map in the vicinity of the Lp locus.

Looptail (Lp) is a mutation that profoundly affects neurulation in mouse and is characterized by craniorachischisis, an open neural tube extending from the midbrain to the tail in embryos homozygous for the mutation. Lp maps to the distal portion of mouse chromosome 1, and as part of a positional cloning approach, we have generated a high-resolution linkage map of the Lp chromosomal region. For this, we have carried out extensive segregation analysis in a total of 706 backcross mice informative for Lp and derived from two crosses, (Lp/+ x SJL/J)F1 x SJL/J and (Lp/+ x SWR/J) F1 x SWR/J. In addition, 269 mice from a (Mus spretus x C57BL/6J)F1 x C57BL/6J interspecific backcross were also used to order marker loci and calculate intergene distances for this region. With these mice, a total of 28 DNA markers corresponding to either cloned genes or anonymous markers of the SSLP or SSCP-types were mapped within a 5-cM interval overlapping the Lp region, with the following locus order and interlocus distances (in cM): centromere--D1Mit110/Atp1 beta 1/Cd3 zeta/Cd3 eta/D1Mit145-D1Hun14/D1Mit15- D1Mit111/D1Mit112-D1Mit114-D1Mit148/D1Mit205+ ++/D1Mit36/D1Mit146/D1Mit147/D1Mit270 / D1Hun13-Fcgr2-Mpp-Apoa2/Fcer1 gamma-Lp-D1Mit149/Spna1/Fcer1 alpha-Eph1-Hlix1/D1Mit62. These studies have allowed the delineation of a maximum genetic interval for Lp of 0.5 cM, a size amenable to physical mapping techniques.

Genetic analysis of innate immunity in resistance to Candida albicans.

Systemic candidiasis is a significant cause of nosocomial infections and the mechanisms of defense against Candida albicans in humans remain poorly understood. Studies in animal models have demonstrated the importance of innate immunity in controlling the response to infection. Although Th1 cytokines have been shown to direct the overall outcome of infection, the precise role of the Th1/Th2 response and, more generally, the adaptive immune response as a whole, in systemic candidiasis, appears to apply mainly to the development of resistance to reinfection. A genetic approach to the identification of host factors regulating pathogenesis and susceptibility to C. albicans infection has been used in humans and in mouse models of infection. Mouse mutants bearing experimentally induced mutations in specific genes have provided a systematic tool for directly assessing the role of individual proteins in C. albicans susceptibility. Inbred mouse strains have been valuable in showcasing the spectrum of naturally occurring variations in initial susceptibility to infection, and type of disease developed. Crosses between resistant and susceptible strains have led to the detection of additional gene effects affecting innate immunity. Of particular interest is the major effect of a naturally occurring loss-of-function mutation in the C5 complement component that has become fixed in many inbred strains. These and other studies have shown that both a functional complement pathway and robust inflammatory response are critical for resistance to C. albicans.

Progesterone receptor synthesis and degradation in MCF-7 human breast cancer cells as studied by dense amino acid incorporation. Evidence for a non-hormone binding receptor precursor.

We have used the technique of density labeling of proteins by biosynthetic incorporation of 2H, 13C, 15N (dense) amino acids to study the synthesis and degradation rates of the progesterone receptor in MCF-7 human breast cancer cells. In cells grown in the absence of progestin, sucrose gradient shift analyses reveal that it takes 17 h for the normal density progesterone receptor levels to be reduced to half the initial value, whereas in the presence of 10 nM of the synthetic progestin [3H]R5020, the receptor turns over more rapidly, such that the normal density R5020-occupied progesterone receptor complexes are reduced to half in 12 h. The accelerated progesterone receptor turnover in the presence of [3H]R5020 reflects increased turnover rates of both the A (Mr-85,000) and B (Mr-115,000) subunits, as determined by sodium dodecyl sulfate gel analyses of dense and light receptors photoaffinity labeled with [3H]R5020. In both control and progestin-exposed cells, the time course of progesterone receptor turnover shows a lag of approximately 6 h after dense (15N, 13C, 2H) amino acid exposure, before dense hormone binding receptor species are seen and before normal density progestin binding activity starts decreasing. Since our evaluations of progesterone receptor depend upon its binding of radiolabeled ligand ([3H]R5020), this lag in the density shift kinetics would be consistent with the presence of a non-hormone binding biosynthetic precursor, from which the hormone-binding form of progesterone receptor is derived. A kinetic model is used to analyze the lag-decay profiles and to determine the rate constants for progesterone receptor synthesis, activation to the hormone-binding form, and degradation.

Antiprogestin-receptor complexes: differences in the interaction of the antiprogestin RU38,486 and the progestin R5020 with the progesterone receptor of human breast cancer cells.

In order to understand the molecular basis for antiprogestin action, we have compared the interaction of the antiprogestin [3H]RU38, 486 (RU486) and the progestin [3H]R5020 with the progesterone receptor (PR). In both MCF-7 and T47D human breast cancer cells, we have observed marked differences in the sedimentation properties of the PR on high salt sucrose gradients: while the R5020-receptor complexes sediment at approximately 4 S (4.4 +/- 0.1 S), the RU486-receptor sediments as a prominent 6 S species as well as a 4 S species. This binding is abolished by excess unlabelled R5020, RU486 or progesterone, but is unaffected by excess unlabelled hydrocortisone or dexamethasone, indicating that both the 4 S and 6 S species represent the PR and not glucocorticoid receptor. Although the relative distribution of 4 S and 6 S forms is not altered by treatment with DNAse or RNAse, exposure to 10 mM thioglycerol or to 3 M urea results in conversion of the 6 S to the 4 S form, suggesting that disulfide bonds and hydrophobic interactions are important in maintaining the integrity of the 6 S form. These findings suggest that the 6 S antiprogestin complex is formed as a result of the interaction of PR units with each other or with a different protein. This change in receptor association state may be an important aspect of the antiprogestin activity of RU486.

Expanded bed adsorption of human serum albumin from very dense Saccharomyces cerevesiae suspensions on fluoride-modified zirconia.

The adsorption of proteins from high cell density yeast suspensions on mixed-mode fluoride-modified zirconia (FmZr) particles (38 to 75 microm, surface area of 29 m(2)/g and density of 2.8 g/cm(3)) was investigated using human serum albumin (HSA) added to Saccharomyces cerevesiae as the model expression host. Because of the high density of the porous zirconia particles, HSA (4 mg/mL) can be adsorbed from a 100 g dry cell weight (DCW)/L yeast suspension in a threefold-expanded bed of FmZr. The expanded bed adsorption of any protein from a suspension containing >50 g DCW/L cells has not been previously reported. The FmZr bed expansion characteristics were well represented by the Richardson-Zaki correlation with a particle terminal velocity of 3.1 mm/s and a bed expansion index of 5.4. Expanded bed hydrodynamics were investigated as a function of bed expansion using residence time distribution studies with sodium nitrite as the tracer. The adsorption of HSA on FmZr exhibited features of multicomponent adsorption due to the presence of dimers. The protein binding capacity at 5% breakthrough decreased from 22 mg HSA/mL settled bed void volume for 20 g DCW/L yeast to 15 mg HSA/mL settled bed void volume for 40 g DCW/L yeast and remained unchanged for the higher yeast concentrations (60 to 100 g DCW/L). However, the batch (or equilibrium) binding capacity decreased monotonically as a function of yeast concentration (20 to 100 g DCW/L) and the binding capacity at 100 g DCW/L yeast was fivefold lower compared with that at 20 g DCW/L yeast. The lower batch binding capacity at high cell concentrations resulted from the adsorption of cells at the surface of the particles restricting access of HSA to the intraparticle surface area. Batch (or equilibrium) and column HSA adsorption results indicated that the adsorption of HSA on FmZr occurred at a time scale that may be much faster than that of yeast cells. The zirconia particles were cleaned of adsorbed HSA and yeast with a total of 1500 to 2000 column volumes (over many cycles) of 0. 25 M NaOH, without any significant effect on the chromatographic performance.

Comparison of fluoride modified zirconia with ceramic hydroxyapatite for preparative scale purification of immunoglobulin from serum albumin.

The utility of 50 microns fluoride modified zirconia particles as an easily cleaned and steam sterilizable low pressure preparative scale stationary phase for immunoglobulin purification was investigated and its performance was compared with 40 microns ceramic hydroxyapatite type II (cHAp II) particles. The equilibrium batch binding capacity of both supports for bovine serum albumin decreases monotonically with increase in the adsorption pH, while that for bovine IgG is independent of pH, in the pH ranges studied. The dynamic binding capacity, as determined by breakthrough analysis, was 29 mg BSA/g zirconia for fluoride modified zirconia and 8.6 mg BSA/g cHAp II for cHAp II. Linear gradient conditions were developed for the separation of BSA and IgG on fluoride modified zirconia. The same separation could be accomplished in a shorter time and with better resolution on cHAp II.

Expanded and packed bed albumin adsorption on fluoride modified zirconia.

The expanded bed characteristics of 75-103microm fluoride-modified zirconia (FmZr) particles synthesized by a fed batch oil emulsion process were investigated. These particles are distinguished from commercially available expanded-bed adsorbents by virtue of their high density (2.8 g/cc) and the mixed mode protein retention mechanism which allows for the retention of both cationic and anionic proteins. The linear velocity versus bed porosity data agree with the Richardson-Zaki relationship with the terminal velocity in infinite medium of 2858.4 cm/h and a bed expansion index of 5.1. Residence time distribution (RTD) studies and bovine serum albumin (BSA) adsorption studies were performed as a function of the height of the settled bed to the column diameter (H:D) ratio and degree of bed expansion with superficial velocities of 440 to 870 cm/h. The settled bed, a 2x expanded bed, and a 3x expanded bed were studied for the H:D ratios of 1:1, 2:1, and 3:1. The dynamic binding capacity (DBC) at 5% breakthrough was low (2-8 mg BSA/mL settled bed) and was independent of the H:D ratio or the degree of bed expansion. The saturation DBC was 32.3 +/- 7.0 mg BSA/mL settled bed. The adsorption-desorption kinetics and intraparticle diffusion for protein adsorption on FmZr (38-75 micrometer) were investigated by studying the packed bed RTD and BSA adsorption as a function of temperature and flow rate. The data show that the adsorption-desorption kinetics along with intraparticle diffusion significantly influence protein adsorption on FmZr. Low residence times ( approximately 0.8 min) of BSA result in a DBC at 5% breakthrough which is 3.5-fold lower compared to that at 6-fold higher protein residence time. At low linear velocity (45 cm/h) the breakthrough curve is nearly symmetrical and becomes asymmetrical and more dispersed at higher linear velocity (270 cm/h) due to the influence of slow adsorption-desorption kinetics and intraparticle diffusion. Bioeng 60: 333-340, 1998.

Nhlh1, a basic helix-loop-helix transcription factor, is very tightly linked to the mouse looptail (Lp) mutation.

Looptail (Lp) is a mutation on the distal portion of mouse Chromosome (Chr) 1 that affects neurulation in mouse and is phenotypically expressed by appearance of an open neural tube along the entire antero-posterior axis of the embryo (craniorachischisis). Nhlh1, a member of the basic helix-loop-helix family of transcription factors, is expressed in the developing neural tube in structures affected by the Lp mutation and has been regionally assigned to the distal part of mouse Chr 1. Using a large panel of looptail animals from an (Lp/+ x SWR/J)F1 x SWR/J segregating backcross progeny, we have determined that Nhlh1 maps very close to Lp, with no recombinant detected in 500 informative animals tested; both map within a 0.6-cM segment defined as D1Mit113/Apoa2/Fcer1 gamma-(0.4 cM)-Nhlh1/Lp-(0.2 cM)-Fcer1 alpha/D1Mit149/Spna1. Nucleotide sequencing of Nhlh1 cDNA clones from wild type (WT) and Lp/Lp embryos failed to identify sequence alterations associated with the mutant phenotype. Southern hybridization of genomic DNA from WT and Lp/Lp embryos failed to identify specific rearrangements at or near the Nhlh1 locus, and Northern RNA blotting and RT-PCR evaluation of Nhlh1 mRNA expression indicated that both the levels and types of Nhlh1 mRNAs produced in WT and Lp/Lp embryos were indistinguishable. These studies suggest that Nhlh1 and Lp are not allelic. Nevertheless, Nhlh1 is the Chr 1 marker most tightly linked to Lp identified to date and can, therefore, be used as an excellent entry probe to clone the Lp region.

Method for construction of a simple laboratory-scale nonwoven filament biocatalytic filter

A method is described for constructing a simple laboratory-scale nonwoven filament biocatalytic filter (FBF) from multilayer acrylic vinyl acetate copolymer coated thread (yarn) potentially useful for investigation of biotransformations using entrapped enzymes or viable microorganisms. The porous structure of a commercial 128 &mgr;m, 279 denier, 100% polyester thread was sealed with a latex coat (precoat), then coated with a latex + sulfanilamide-azocasein mixture, and finally coated with a latex top coat to generate the multilayer coated thread. The FBF was constructed by drawing the thread across a 4 cm diameter rubber O-ring in the form of multiple layers and sealing the layers with silicone sealer. The FBF is combatible with a commercially available filter housing containing a flow distrtibutor. A method for characterizing the permeability of the sealant (top) coat of the FBF using azocasein release was also developed.