Participation of membrane progesterone receptor α in the inhibitory effect of progesterone on prolactin secretion.

The membrane progesterone receptors (mPRα, mPRß, mPRγ, mPRδ and mPRε) are known to mediate rapid nongenomic progesterone functions in different cell types. However, the functions of these receptors in the pituitary have not been reported to date. In the present study, we show that the expression of mPRα was the highest among the mPRs in the rat anterior pituitary gland. Immunostaining of mPRα was detected in somatotrophs, gonadotrophs and lactotrophs. Interestingly, 63% of mPRα-positive cells within the pituitary were lactotrophs, suggesting that mPRα is involved in controlling prolactin (PRL) secretion in the pituitary. To test this hypothesis, rat pituitaries were incubated (1 hour) with either progesterone (P4) or the mPRα-specific agonist Org OD 02-0. PRL secretion was then measured by radioimmunoassay. The results of this experiment revealed that both P4 and Org OD 02-0 decreased PRL secretion. Moreover, the results from the GH3 cell line (CCL-82.1) showed that P4 and Org OD 02-0 inhibited PRL release, although the nuclear PR agonist R5020 was ineffective. Our investigation of the cellular mechanisms behind mPRα activity indicated that both P4 and Org OD 02-0 decreased cAMP accumulation, whereas R5020 was ineffective. In addition, the Org OD 02-0-effect on PRL release was blocked by pretreatment with pertussis toxin, an inhibitor of Go/Gi proteins. Because transforming growth factor (TGF)ß1 is a potent inhibitor of PRL secretion in lactotrophs, we lastly evaluated whether TGFß1 was activated by progesterone and whether this effect was mediated by mPRα. Our results showed that P4 and Org OD 02-0, but not R5020, increased active TGFß1 levels. This effect was not observed when cells were transfected with mPRα-small interfering RNA. Taken together, these data provide new evidence suggesting that mPRα mediates the progesterone inhibitory effect on PRL secretion through both decreases in cAMP levels and activation of TGFß1 in the lactotroph population.

SIV vpx is essential for macrophage infection but not for development of AIDS.

Analysis of rhesus macaques infected with a vpx deletion mutant virus of simian immunodeficiency virus mac239 (SIVΔvpx) demonstrates that Vpx is essential for efficient monocyte/macrophage infection in vivo but is not necessary for development of AIDS. To compare myeloid-lineage cell infection in monkeys infected with SIVΔvpx compared to SIVmac239, we analyzed lymphoid and gastrointestinal tissues from SIVΔvpx-infected rhesus (n = 5), SIVmac239-infected rhesus with SIV encephalitis (7 SIV239E), those without encephalitis (4 SIV239noE), and other SIV mutant viruses with low viral loads (4 SIVΔnef, 2 SIVΔ3). SIV+ macrophages and the percentage of total SIV+ cells that were macrophages in spleen and lymph nodes were significantly lower in rhesus infected with SIVΔvpx (2.2%) compared to those infected with SIV239E (22.7%), SIV239noE (8.2%), and SIV mutant viruses (10.1%). In colon, SIVΔvpx monkeys had fewer SIV+ cells, no SIV+ macrophages, and lower percentage of SIV+ cells that were macrophages than the other 3 groups. Only 2 SIVΔvpx monkeys exhibited detectable virus in the colon. We demonstrate that Vpx is essential for efficient macrophage infection in vivo and that simian AIDS and death can occur in the absence of detectable macrophage infection.

An efficient synthesis of dopamine transporter tracer [¹8F]FECNT.

A simple synthesis of the dopamine transporter ligand [(18)F]FECNT with high radiochemical yield and short synthesis time, suitable for routine production is reported. Reaction of 2ß-carbomethoxy-3ß-(4-chlorophenyl)nortropane with [(18)F]2-fluoroethyl triflate ([(18)F]FEtOTf) at room temperature for 4 min provided [(18)F]FECNT in 84% decay corrected radiochemical yield. Since [(18)F]FEtOTf was prepared from [(18)F]2-fluoroethyl bromide that was isolated from its starting material, formation of unwanted side products and the amount of expensive precursor used could be greatly reduced. The overall radiochemical yields of [(18)F]FECNT were 40% (n=29) and the total synthesis time was ca. 100 min. The average specific activity of [(18)F]FECNT was 377.4 GBq/µmol (10.2 Ci/µmol).

18F-labeled resin microspheres as surrogates for 90Y resin microspheres used in the treatment of hepatic tumors: a radiolabeling and PET validation study.

(90)Y-labeled resin microspheres (SIR-Spheres) are currently used to treat patients with primary and metastatic solid liver tumors. This treatment is typically palliative since patients have exhausted all other standard treatment options. Improving the quality of life and extending patient survival are typical benchmarks for tracking patient response. However, the current method for predicting microsphere biodistributions with (99m)Tc-labeled macroaggregated albumin (MAA) does not correlate well with patient response. This work presents the development of a new (18)F-labeled resin microsphere to serve as a surrogate for the treatment microsphere and to employ the superior resolution and sensitivity of positron emission tomography (PET). The (18)F microsphere biodistributions were determined in a rabbit using PET imaging and histological review. The PET-based uptake ratio was shown to agree with the histological findings to better than 3%. In addition, the radiolabeling process was shown to be rapid, efficient and relatively stable in vivo.

Aromatic L-amino acid decarboxylase turnover in vivo in rhesus macaque striatum: a microPET study.

The aromatic L-amino acid decarboxylase (AAAD) is involved in the de novo synthesis of dopamine, a neurotransmitter crucial in cognitive, neurobehavioral and motor functions. The goal of this study was to assess the in vivo turnover rate of AAAD enzyme protein in the rhesus macaque striatum by monitoring, using microPET imaging with the tracer [(18)F]fluoro-m-tyrosine (FMT), the recovery of enzyme activity after suicide inhibition. Results showed the AAAD turnover half-life to be about 86 h while total recovery was estimated to be 16 days after complete inhibition. Despite this relatively slow AAAD recovery, the animals displayed normal movement and behavior within 24 h. Based on the PET results, at 24 h, the animals have recovered about 20% of normal AAAD function. These findings show that normal movement and behavior do not depend on complete recovery of AAAD function but likely on pre-synaptic and post-synaptic compensatory mechanisms.

Production of [17F]CH3F (t1/2=65 s), an improved PET tracer for rCBF measurement.

Production of 17F (t1/2=65 s) in the form of [17F] F2 has been achieved using both the 20Ne(p,alpha)17F and 16O(d,n)17F reactions with 11 MeV protons and 6 MeV deuterons, respectively. Yields have proven suitable for subsequent radiosynthesis of the blood flow tracer, [17F]CH3F (>60 mCi in saline), currently in use for fast repetition human studies of regional cerebral blood flow with positron emission tomography. Thick target yields of 15 mCi /microA for protons and 44 mCi/microA for deuterons have been measured for [17F]F2.

Water-cooled grid support for high-power irradiation with thin target windows.

A new thin window support system for the accelerator production of positron emitters (e.g. 17F, 18F 11C, 15O) has been developed. The integrated support grid and cooling design has been optimized for 6-13 MeV protons or deuterons. The water-cooled support grid regularly operated at > 100 microA of 6 MeV deuterons and protons. The grid performed without failure at > or = 50 microA of 13 MeV protons on a 3.1 MPa gas target using 25.4 microm aluminum target foil. Transmission for the smallest hole grid of 72% based on uniform parallel beam agreed with the measured yield of 71 +/- 1% compared to the theoretical maximum yield.

Simulation of a cross-flow shrinking-bed reactor for the hydrolysis of lignocellulosics.

An idealized model is developed for the case in which biomass slurry is conveyed through an annulus, with water or steam entering through an inner porous wall and liquid product leaving through an outer porous wall. It is assumed that the ratio of occluded liquid to solid in the slurry is a constant, Rws, and that non-occluded water is immediately removed from the reactor. The goal of > 90% sugar yield with > 10% sugar in the product is almost reached (88% glucose yield, 91% xylose yield, 47 g/l glucose and 45 g/l xylose) at 240 degrees C, 1% acid. Rws = 1 and a radial wash water flow of three times the initial mass flow of solids to the reactor per meter of reactor length per g/l of sugar concentration in the occluded water. If Rws is limited to 3, the yield falls to 85% and the total sugar concentration to 61 g/l. Even without cross-flow wash, the yields can be increased by about 16 percentage points, compared to plug flow, by extracting excess liquid through the outer wall as it is formed. At 200 degrees C, where one might prefer to operate for ease of control and concern about the possibility of making fermentation inhibitors at higher temperatures, the maximum glucose yield in a plug-flow reactor is low (12-13%) whereas in a cross-flow reactor, at a high cross-flow wash rate, it can still be quite high (60-83%) but at a very low concentration (0.57-1.47%). In these simulations it is assumed that one-half of the inerts is solubilized. The formation of oligomers is neglected.

Substrate reactivity as a function of the extent of reaction in the enzymatic hydrolysis of lignocellulose.

In an effort to better understand the role of the substrate in the rapid fall off in the rate of enzymatic hydrolysis of cellulose with conversion, substrate reactivity was measured as a function of conversion. These measurements were made by interrupting the hydrolysis of pretreated wood at various degrees of conversion; and, after boiling and washing, restarting the hydrolysis in fresh buffer with fresh enzyme. The comparison of the restart rate per enzyme adsorbed with the initial rate per enzyme adsorbed, both extrapolated back to zero conversion, provides a measurement of the substrate reactivity without the complications of product inhibition or cellulase inactivation. The results indicate that the substrate reactivity falls only modestly as conversion increases. However, the restart rate is still higher than the rate of the uninterrupted hydrolysis, particularly at high conversion. Hence we conclude that the loss of substrate reactivity is not the principal cause for the long residence time required for complete conversion. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 650-655, 1997.

An enhancer in the first intron of the human purine nucleoside phosphorylase-encoding gene.

In previous studies characterizing intron-dependent expression of the human purine nucleoside phosphorylase-encoding gene (PNP), we identified a putative enhancer sequence in the first intron which was capable of mediating increased cat reporter gene expression in transfected murine NIH 3T3 cells in a position- and orientation-independent manner. In order to further characterize this enhancer activity, the nucleotide sequence was determined for the region of intron 1 to which this activity was originally ascribed. The sequence was analyzed for the presence of binding sites for known transcription factors, but none were identified. A 444-bp downstream portion of the intron-1 sequence enhanced cat expression either in conjunction with a human PNP promoter sequence or with a 105-bp heterologous herpes simplex virus thymidine kinase (TK) promoter. Nested deletions of the downstream intron-1 sequence fused to a TK::cat fusion gene localized the enhancer activity to a 170-bp sequence in intron 1. A 154-bp HgiAI fragment (bp 424 to 577 of intron 1) excised from this region contained enhancer activity which varied directly with the number of fragments inserted upstream from the TK::cat fusion gene. However, inversion of the HgiAI fragment in a PNP abbreviated gene, or relocation of the HgiAI fragment from intron 1 to a position upstream from the PNP promoter, reduced or eliminated PNP expression. The effect of the intron-1 enhancer element on PNP expression is thus maximized in a position- and orientation-dependent manner.

A synergistic kinetics model for enzymatic cellulose hydrolysis compared to degree-of-synergism experimental results.

It is demonstrated that a two-enzyme component synergistic model can account for the observation that the degree of synergism goes through a maximum as the total enzyme concentration is increased. The degree of synergism is low at low enzyme concentration because the extent of conversion is low and therefore the cellulose chain ends, present originally, are not exhausted; thus the action of the cellobiohydrolase (CBH) is not dependent on the chain ends generated by the endoglucanase (EG). The degree of synergism declines at high enzyme concentration due to saturation of adsorption sites with CBH, thus decreasing the generation of chain ends by EG.

Purging of small cell lung cancer cells from bone marrow using immunomagnetic beads and a flow-through device.

Immunomagnetic beads can be used to remove subpopulations of cells from a mixed cell suspension in a flow-through system. One application of this process is the removal of tumor cells from bone marrow prior to its use in autologous bone marrow transplantation (ABMT). Based on preliminary data showing that three monoclonal antibodies (MoAb) (SCCL 175, HNK-1, and TFS-4) gave optimal separation in small-scale experiments, we have designed a large-scale separator suitable for clinical use. In our separator, the cell suspension flows through a 150 ml baffled transfer pack which is held over an array of permanent magnets. Direct (one MoAb only) and indirect (MoAb and anti-mouse antibody) methods of binding beads to cells were investigated as were the effects of temperature, bead to cell ratio, and medium additives on tumor cell removal and normal cell recovery. We determined the optimal separation conditions to be the indirect method of binding at 22 degrees C using a bead to tumor cell ratio of 25:1. Testing of the device on DMS-273 small cell lung cancer (SCCL) cells mixed with normal human bone marrow mononuclear cells resulted in a mean tumor cell removal of 3.64 logs (99.977%) with a concomitant mean normal granulocyte-monocyte colony forming unit (CFU-GM) recovery of 61.3%. These experiments form the basis to use the immunomagnetic beads to purify bone marrow from patients with SCCL for use in ABMT.

Adsorption of cellulase from Trichoderma reesei on cellulose and lignacious residue in wood pretreated by dilute sulfuric acid with explosive decompression.

The adsorption of cellulase on cellulose and a lignacious residue was examined by using cellulase from Trichoderma reesei, hardwood pretreated by dilute sulfuric acid under high pressure, and a lignacious residue prepared by a complete enzymatic hydrolysis of the pretreated wood. A significant amount of cellulase was found to adsorb on the lignacious residue during the hydrolysis of the pretreated wood. Hence, the adsorption of enzyme on the lignacious residue as well as cellulose must be taken into account in the development of the hydrolysis kinetics. It was found that the adsorption of enzyme on cellulose and on the lignacious residue could be represented by Langmuir type isotherms. The data show that the pretreatment at a higher temperature results in more enzyme adsorption on the cellulose fraction and less on the lignacious residue fraction. The relationship between the hydrolysis rate and the amount of enzyme adsorbed is discussed.

Effect of pore size in substrate and diffusion of enzyme on hydrolysis of cellulosic materials with cellulases.

The effect of cellulase size on hydrolysis was studied by comparing the behavior of crosslinked cellulase (CC) with normal cellulase (FC). The average molecular weight of the CC was at least three times the molecular weight of the FC. The amounts of each enzyme were adjusted so that the degree of solubilization after 2 h was the same. The degree of solubilization of Avicel with CC was higher than that with FC in the late stage of reaction. The degree of solubilization of pretreated lignocelluloses was much greater than that of Avicel, but the degree of solubilization with CC was lower than that with FC at all times during the reaction. The degree of solubilization of artificial lignified Avicel was higher with FC than with CC, but the degree of solubilization of de-lignified the artificial lignified Avicel was lower with FC than with CC. The degree of solubilization of amorphous cellulose with FC was the same as that with CC at all times during the reaction. These behaviors are examined by the hypothesis that when small pores dominate, the smaller enzyme components diffuse into the pores and become inactive since synergism with the larger components is no longer possible, whereas, when larger pores dominate, the entire enzyme can diffuse in and therefore the available surface area is increased. This hypothesis is supported by direct measurement of the pore size in two of the substrates and by diffusion inside Avicel of only smaller molecular cellulase component.

A model of enzyme adsorption and hydrolysis of microcrystalline cellulose with slow deactivation of the adsorbed enzyme.

Reduction in the activity and the concentration of the adsorbed enzyme are noted in the experimental data. Two alternative mechanisms, inactivation of the adsorbed enzyme and mass transfer of the enzyme from the bulk solution to the solution within the cellulose fibril where the cellulase is assumed to be inactive, are used to represent the decline in activity. The decline in concentration of the adsorbed enzyme is represented by a modest product inhibition and, more importantly, the assumption that the concentration of the adsorption sites is proportional to the square of the remaining substrate concentration. Measurements of both adsorbed enzyme and product concentration over time are used in determining parameter values. The model is applied to a series of experiments having a 10-fold range of substrate concentration and to an experiment in which the product is removed continuously. For both deactivation mechanisms, a very good representation of product concentration (standard deviation 3.6%) is obtained over the full period (168 h) of hydrolysis; the representation of adsorbed enzyme is, however, less accurate (standard deviation 6.7-6.8%).

Kinetics of cellobiose hydrolysis using cellobiase composites from Ttrichoderma reesei and Aspergillus niger.

The enzymatic hydrolysis of cellulose to glucose involves the formation of cellobiose as an intermediate. It has been found necessary(1) to add cellobiase from Aspergillus niger (NOVO) to the cellobiase component of Trichoderma reesei mutant Rut C-30 (Natick) cellulase enzymes in order to obtain after 48 h complete conversion of the cellobiose formed in the enzymatic hydrolysis of biomass. This study of the cellobiase activity of these two enzyme sources was undertaken as a first step in the formation of a kinetic model for cellulose hydrolysis that can be used in process design. In order to cover the full range of cellobiose concentrations, it was necessary to develop separate kinetic parameters for high- and low-concentration ranges of cellobiose for the enzymes from each organism. Competitive glucose inhibition was observed with the enzymes from both organisms. Substrate inhibition was observed only with the A. niger enzymes.

A comparative study of the enzymatic hydrolysis of acid-pretreated white pine and mixed hardwood.

Removal of hemicellulose by acid pretreatment in a flow reactor followed by enzymatic hydrolysis of the neutralized slurry has resulted in glucose yields as high as 95% for mixed hardwood. For white pine, however, the maximum glucose yield is 65%. Although pine has a higher extractives content, removal of the extractives prior to enzymatic hydrolysis does not increases the glucose yield. Pore size measurements reveal that the increase in pore volume, in the size range of the cellulase molecule, following pretreatment for pine is only about one-half the value obtained with mixed hardwood. This suggests that pore volume is an important determinant of substrate-enzyme reactivity.