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Postsynaptic receptors in bipolar cells were studied by focal application of glutamate and GABA to the outer and inner plexiform layers (OPL and IPL) under visual guidance in living retinal slices of the tiger salamander. Two different types of conductance change could be elicited in bipolar cells by applying glutamate to the OPL. In off-center cells, which had axon telodendria ramifying in the distal 55% of the IPL, glutamate elicited a conductance increase associated with a reversal potential near -5 mV. In on-center cells, which had telodendria stratified in the proximal 45% of the IPL, glutamate caused a conductance decrease associated with a reversal potential near -11 mV. These observations suggest that glutamate gates relatively nonspecific cation channels at synapses between photoreceptors and bipolar cell dendrites. Application of glutamate to the IPL elicited no conductance change in Co2+ Ringer's solution, but in normal Ringer's it generated a conductance increase associated with a reversal potential near the chloride equilibrium potential (ECl). These findings are consistent with the notion that glutamate receptors exist in GABAergic and/or glycinergic amacrine cells, and that glutamate in the IPL depolarizes these cells, causing GABA and/or glycine release and the opening of chloride channels in bipolar cell axon terminals. In Co2+ Ringer's, application of GABA at the OPL elicited no conductance changes in bipolar cells, suggesting that GABA receptors do not exist on bipolar cell dendrites. Applied at the IPL, GABA elicited large conductance increases associated with a reversal potential near ECl. Implications of these results for the functional circuitry of the tiger salamander retina are discussed.

1. Rods, cones, and horizontal cells (HCs) were recorded in superfused, flat-mounted isolated retinas of the larval tiger salamander, Ambystoma tigrinum, under dark- and light-adapted conditions. 2. Under dark-adapted conditions, HC responses to dim 500-nm light stimuli were mediated only by rods. In the linear voltage range (near the dark potentials), the average response to a light step of 0.5 s (500 nm, 0.438 photons per micron2 per s) was 0.41 +/- 0.06 (SD) mV for rods and 1.86 +/- 0.52 mV for HCs. The step sensitivity of rods was approximately 0.94 mV per photon micron2 s, or 0.032 mV per activated rhodopsin molecule (Rh*) rod second, and the step sensitivity of HCs was approximately 4.25 +/- 1.19 mV per photon micron2 s or 0.14 +/- 0.04 mV per Rh* rod second. The chord voltage gain of the rod-HC synapse had an average value of 4.54 and a range from 2.68 to 7.32. 3. By the use of the spectral subtraction method, we found that the average cone-mediated HC response to a 750-nm light step that elicited an average cone response of 0.73 +/- 0.20 mV was 1.15 +/- 0.31 mV. The step sensitivity of cones under dark-adapted conditions was 0.0012 mV per photon micron2 s, and that of the cone-mediated-HC response was 0.0019 mV per photon micron2 s. The chord voltage gains of the cone-HC synapses under dark-adapted conditions had an average value of 1.58 and a range from 0.82 to 2.05. 4. Under light-adapted conditions (with a 500-nm/-2.40 background light, which desensitized rod responses but did not substantially reduce the cone responses), the cones had an average response to a light step of 0.5 s (500 nm/-3.3) of 0.78 +/- 0.09 mV, and this response did not vary with time. The HC response to the same light step had an average value of 3.95 +/- 3.41 mV 3 min after the background light onset, and it increased with time until reaching a steady-state value of 5.95 +/- 3.63 mV approximately 15 min after the background light onset. The average chord voltage gain of the cone-HC synapse under such light-adapted conditions was 5.06 at 3 min after background light onset and 7.63 at 15 min after background light onset. These values are approximately 3-5 times higher than the chord voltage gain of the cone-HC synapse under dark-adapted conditions. 5. The background-induced increase of the chord voltage gain of the cone-HC synapse suggests that similarly to the rod-HC synapse, the voltage gain of the cone-HC synapse in the tiger salamander retina can also be modulated by light. Additionally, our results suggest that certain time-dependent process(es) in the synaptic cleft or postsynaptic membrane may be responsible for such modulation. 6. In addition to determining the average values of response sensitivity and chord voltage gains of the rod- and cone-HC synapses, we studied the variation of these parameters among different HCs. HCs with higher rod-HC synaptic gain had lower cone-HC synaptic gain, and HCs with lower rod-HC synaptic gain exhibited higher cone-HC synaptic gain under both dark- and light-adapted conditions. This suggests that the rod-HC and cone-HC synaptic gains in HCs are complementary to each other, and voltage responses of all HCs under dark- or light-adapted conditions are of comparable amplitudes.

The effect of an arterial filter on visceral emboli was quantified with autologous indium-111 labeled platelets (INPLT) during cardiopulmonary bypass (CPB) in Yorkshire pigs. Biodistribution of INPLT was determined in 12 control pigs (30-35 kg, unoperated control [n = 6] and sham operated control [n = 6]). CPB was carried out with (n = 6) and without (n = 6) an arterial filter in 12 pigs at a flow rate of 2.5-3.5 L/min. Platelets labeled with In-111 tropolone (650-780 microCi) were injected intravenously 24 hr before CPB. All pigs were systemically heparinized (activated coagulation time > 400 sec); CPB was instituted with a roller pump, an extraluminal blood flow oxygenator (Bentley Univox, 1.8 m2), and an arterial filter (0.25 m2) and continued for 3 hr. Platelet kinetics, pooling, and counts were monitored by a Geiger probe and a Coulter counter. The thrombi in the oxygenator and arterial filter and emboli in viscera and brain were imaged with a gamma camera and measured with an ion chamber and gamma counter. Percentage of INPLT (mean +/- SD) in organs, tissues, and components of the circuit in four groups of pigs was calculated. Flow cytometry with antibodies to CD61 (GPIIIa) and CD62P (GMP-140: control) of porcine platelets was carried out with blood samples taken before, during, and after CPB for estimation of circulating platelet aggregates and platelet microparticles. Pulmonary, renal, cardiac, and cerebral emboli in pigs undergoing CPB with and without a filter were similar (p < 0.1). The amount of filter adherent thrombi was small (0.04 +/- 0.01%); oxygenator adherent thrombus in both groups was similar (p < 0.1). Emboli were found in the cerebral medulla, hippocampus, and posterior cerebral cortex in both groups. During CPB, the arterial filter functioned minimally as a trap for platelet thrombi detached from the oxygenator and circulating emboli. Flow cytometry of blood demonstrated the shift of equilibria from single platelets to platelet aggregates and microparticles during CPB and their gradual reversal to single platelets after CPB; the loosely adherent emboli disaggregated and further shifted these equilibria to single platelets and smaller aggregates, probably through the action of endogenous nitric oxide and prostacyclin. The emboli were trapped in organs and tissues and microparticles were sequestered by the reticuloendothelial system.

The propeptide sequences of the vitamin K-dependent clotting factors serve as a recognition site for the enzyme gamma-glutamylcarboxylase, which catalyzes the carboxylation of glutamic acid residues at the NH2 terminus of the mature protein. We describe a mutation in the propeptide of Factor IX that results in warfarin sensitivity because of reduced affinity of the carboxylase for the Factor IX precursor. The proband has a Factor IX activity level of > 100% off warfarin and < 1% on warfarin, at a point where other vitamin K-dependent factors were at 30-40% activity levels. Direct sequence analysis of amplified genomic DNA from all eight exons and exon-intron junctions showed a single guanosine-->adenosine transition at nucleotide 6346 resulting in an alanine to threonine change at residue -10 in the propeptide. To define the mechanism by which the mutation resulted in warfarin sensitivity, we analyzed wild-type and mutant recombinant peptides in an in vitro carboxylation reaction. The peptides that were analyzed included the wild-type sequence, the Ala-10-->Thr sequence, and Ala-10-->Gly, a substitution based on the sequence in bone gamma-carboxyglutamic acid protein. Measurement of C02 incorporation at a range of peptide concentrations yielded a Vmax of 343 cpm/min/reaction for the wild-type peptide, and Vmax values of 638 and 726 for A-10T and A-10G respectively, a difference of only twofold. The Km values, on the other hand, showed a 33-fold difference between wild-type and the variants, with a value of 0.29 microM for wild-type, and 10.9 and 9.50 microM, respectively, for A-10T and A-10G. Similar kinetic experiments showed no substantial differences between wild-type and mutant peptides in kinetic parameters of the carboxylase-peptide complexes for reduced vitamin K. We conclude that the major defect resulting from the Factor IX Ala-l0-->Thr mutation is a reduction in affinity of the carboxylase for the mutant propeptide. These studies delineate a novel mechanism for warfarin sensitivity. In addition, the data may also explain the observation that bone Gla protein is more sensitive to warfarin than the coagulation proteins.

Nitric oxide generation by L-arginine (2 mg/kg/min) infusion during cardiopulmonary bypass (CPB) increases blood flow to all organs and reduces cytokine induced organ damage by reducing the level of marginating neutrophils (Ns). The N-trapping in the oxygenator (OX), arterial filter (AF), cardiotomy reservoir (CR), and N-margination were quantified with indium 111 labeled autologous neutrophils (INN) in nine groups of 40 Yorkshire pigs (30-35 kg). Cardiopulmonary bypass (180 min or 90 min CPB, 90 min reperfusion) was carried out at 2.5-3.5 L/min and at two temperatures (18 degrees C, 28 degrees C). The INN (650-780 microCi) was administered intravenously 15 mins before CPB. All pigs received heparin systemically (activated coagulation time > 400 secs); CPB was instituted with a roller pump, OX (Univox 1.8 m2), AF (0.25 m2), and CR (BCR-3500, Bentley Lab, Irvine, CA). The INN distribution in the device (OX, AF, CR) and organs was imaged with a gamma camera and measured with an ion chamber and a gamma counter. The LA infusion decreased N-trapping, estimated as the percent of injected INN (mean +/- standard deviation), in OX from control (2.7 +/- 2.02)% to (0.94 +/- 0.29)%, and margination in lung from control (48 +/- 4)% to minimal levels (23 +/- 2)% (p < 0.01). In the CPB reperfusion group, a beneficial effect was observed at LA low dose and toxicity of higher N-margination at 15 mg/ kg/min. Neither CPB temperature nor Leumedin affected N-margination significantly.

To understand the allele frequency of the G1691A mutation of the coagulation factor V gene (factor V Leiden) in Chinese, 618 Chinese individuals, including 54 cases with venous thrombosis, were analyzed. Only one case in the control group was heterozygous for the 1691G allele and the 1691A allele. Our data suggest that the factor V Leiden is rare in Chinese.

The human LH receptor (hLHR) is a member of the G protein-coupled receptors characterized by the presence of seven-transmembrane (TM) helices. Inactivating mutations of the hLHR lead to Leydig cell hypoplasia (LCH), a form of male pseudohermaphroditism resulting from the failure of fetal testicular Leydig cell differentiation. We have identified three mutations of the hLHR in a patient with LCH: deletion of exon 8 (delta Exon 8), A872G transition resulting in Asn291Ser substitution in the extracellular domain, and C1847A transversion resulting in Ser616Tyr substitution in the seventh TM helix. Nucleotide sequencing, gene dosage, and allele-specific amplification analyses revealed that exon 8 deletion and the two missense mutations are present in different alleles of the hLHR. Constructs of mutated hLHR (hLHR-delta Exon8, hLHR-872/1847, hLHR-1847, and hLHR-872) were used to transfect 293 cells, and the properties of the hLHR expressed were examined. Ligand-binding assays failed to detect the expression of hLHR-delta Exon8. Transfectants expressing hLHR-872/1847 demonstrated greatly reduced ligand binding and ligand-induced cAMP accumulation in comparison to those expressing wild type hLHR. Similar reduction in cAMP accumulation was observed in transfectants expressing hLHR-1847, but not hLHR-872 alone. These findings suggest that, in addition to the 7-TM helices, the polypeptide encoded by exon 8 plays an important role in LHR expression and signal transduction. On the other hand, glycosylation of Asn291 may not be critical for these activities. These results also establish that LCH can result from impaired signal transduction due to compound heterozygous mutations. Implications of these mutations on structure-function relationship of the hLHR and the genotype-phenotype correlation in LCH are discussed.

Male-limited precocious puberty (MPP) is a gonadotropin-independent disorder that occurs sporadically or is inherited in an autosomal dominant, male-limited pattern. Recent studies have identified constitutively activating missense mutations in the human luteinizing hormone receptor (hLHR) gene leading to Leydig cell activation and precocious puberty. Patients with sporadic MPP (SMPP) or with different ethnic backgrounds appear to have a greater likelihood of having novel mutations. In the current study we examined genomic DNA from two unrelated cases of SMPP of African-American descent for novel mutations of the hLHR gene. A heterozygous A to C transversion at nucleotide 1723 resulting in substitution of Leu for lle575 in transmembrane helix 6 was identified. Human embryonic kidney cells transfected with cDNA for the mutant hLHR-I575L, created by polymerase chain reaction-based mutagenesis of the wild-type (hLHR-wt) cDNA, exhibited increased basal levels of cAMP production in the absence of agonist, indicating constitutive activation. Surface expression of hLHR-I575L, as reflected by human chorionic gonadotropin binding, was diminished compared to hLHR-wt, while agonist affinity was unaffected. With the exception of two polymorphic bases, no mutation was identified within the coding sequence of the hLHR in the second case of SMPP. We conclude that I575L is a unique constitutively activating mutation that impairs cell surface expression of the receptor but does not alter agonist affinity. Furthermore, mutations of the hLHR gene causing SMPP are highly heterogeneous and may be found in regions other than exon 11 of the hLHR. Last, patients with MPP from different ethnic backgrounds are likely to have novel mutations.

Purpose: To maximize the iontophoretic transdermal delivery rate of thyrotropin-releasing hormone (TRH) facilitated by periodically monophase-pulsed current across excised skin., Methods: The pH of the buffer, the ionic strength in the solution, the frequency of the periodically monophase-pulsed current and the current on/off ratio were chosen as the key variables. A response surface method was applied to optimize the transdermal delivery rate of TRH under different operational conditions., Results: The optimum operating conditions were achieved via experimentation based on the response surface method by systematically adjusting the pH of the buffer, the ionic strength in the solution, the current amplitude, frequency and the active temporal ratio of the pulsed current. The rate of permeation of TRH crossing the skin during iontophoresis varied from two to ten-fold, depending on operating conditions., Conclusions: Only a few steps, two in this work, were needed to reach the optimal. The response surface near the region of the maximal point was thoroughly described with a quadratic function. A maximal transdermal rate of permeation of TRH, 103.2 micrograms h-1 cm-2, was obtained when the donor solution was at pH = 7.0, ionic strength = 0.037, and with a periodically monophase-pulsed current iontophoresis with duty cycle = 75%. The effect of pulse frequency was not statistically significant.

The effect of positioning on pulmonary function has been previously evaluated, and the prone position has been reported to be preferable for neonates with various respiratory diseases. Studies in healthy neonates have yielded conflicting results. Using a crying pulmonary function test, we examined the effect of positioning on pulmonary function in healthy full-term neonates. Thirty-nine infants with a mean birthweight (+/- SD) of 3,140 +/- 379 g and a mean gestational age (+/- SD) of 39.8 +/- 1.6 weeks were investigated during the first 6 hours of life. Measurements were obtained in both supine and prone positions using a computerized volume-flow system. There were statistically significant decreases in crying vital capacity (CVC) and peak expiratory flow rate (PEF) in the prone compared with the supine position. However, there were no significant differences in forced expiratory flow rate at 75% (V(75)), 50% (V(55)), and 25% (V(25)) of vital capacity between the two positions. These results suggest that prone positioning decreases lung volume and increases resistance of upper airways. We conclude that healthy neonates should be in the supine posture for optimal ventilation.

Low-density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor is a member of the low-density lipoprotein receptor family. It is known to bind a wide variety of unrelated ligands including alpha 2-macroglobulin-proteinase complexes, tissue plasminogen activator, apolipoprotein E-enriched very low density lipoprotein, lipoprotein lipase, and Pseudomonas exotoxin A. Receptor-associated protein (RAP), a protein which copurifies with LRP, can inhibit the binding and internalization of all known ligands to LRP. Recent studies have shown that some ligands can bind to more than one receptor in this family. However, the ability of low-density lipoprotein (LDL) to bind to LRP in addition to the LDL receptor has not been demonstrated consistently. In this study we demonstrate that LDL binds with high affinity to macrophage cell surface receptors at 4 degrees C (Kd = 1.8 nM) and competes for the binding of a receptor-recognized form of alpha 2-macroglobulin (alpha 2M*) (Ki = 3 nM). alpha 2M* and RAP can inhibit the binding of LDL to macrophages completely (96 and 100% inhibition, respectively), after cell surface heparin has been removed by treatment with heparinase. Using a solid-phase assay, we show that LDL binds specifically, saturably, and with high affinity to purified LRP (Kd = 5 nM). LDL can also completely inhibit the binding of alpha 2M* to purified LRP. These results indicate that LDL binds directly to LRP. The ability of LDL to cross-compete with alpha 2M* for binding to LRP suggests that LDL binds to a similar or overlapping site as alpha 2M*. In addition, the ability of alpha 2M* to inhibit most of the receptor-mediated binding of LDL to macrophages suggests that LDL receptors on murine peritoneal macrophages are predominantly LRP.

We report here the design, construction and testing of a self-inactivating (Sin) retrovirus promoter-trap vector suitable for identifying and isolating transcriptionally active regions from the mouse genome. When this vector, which contains the bacterial aph gene as its reporter, is integrated into a site downstream from an active host cell promoter, it expresses aph, whose product, aminoglycoside phosphotransferase, produces resistance to the antibiotic G418 in mammalian cells. The construct also contains a native aph promoter which functions in bacteria, but not in mouse cells, to express kanamycin (Km) resistance, plus an adjacent pBR322-derived replication origin. Thus, mammalian DNA segments containing actively transcribed regions flanking aph can be quickly isolated by restriction endonuclease treatment of total DNA from provirus-containing mouse cells, followed by self-ligation, transformation and Km selection of plasmids carried by bacteria transformed with this DNA. We tested this Sin retrovirus promoter-trap system by isolating eight DNA segments upstream to the provirus integration sites in the genome of virus-infected mouse F9 cells. We found that the Sin retrovirus vector produces a high yield of infectious virus particles carrying aph, and that the isolated genomic DNA fragments of F9 cells are transcriptionally active.

Leydig cell hypoplasia (LCH) is a form of male pseudohermaphroditism in which Leydig cell differentiation and testosterone production are impaired. This report describes the first case of a nonsense mutation (A1635C) in exon 11 of the human luteinizing hormone receptor (hLHR) gene in two sisters with LCH. This mutation causes loss of function of the receptor by introducing a stop codon at residue 545 in transmembrane helix 5 of the hLHR. Surface expression of the truncated hLHR (hLHR-t545) in human embryonic kidney cells stably transfected with cDNA encoding hLHR-t545 was diminished compared to the wild-type hLHR and hCG-induced cAMP accumulation was impaired. These results establish that single base mutations in exon 11 of the hLHR gene can produce inactivation as well as activation of the hLHR. Furthermore, they demonstrate that functional domains between transmembrane helix 5 and the C-terminal cytoplasmic tail of the hLHR are required for normal cell surface expression of the receptor and signal transduction.

A monoclonal antibody against human DNA polymerase delta (pol delta) was isolated with properties suitable for its utilization for immunoaffinity chromatography. The antibody was immobilized after periodate oxidation and coupled to a hydrazide-activated support. Starting from a partially purified preparation, calf thymus pol delta was purified about 200-fold in a single step. Further purification on ssDNA-cellulose resulted in isolation of a homogeneous preparation. The amount of enzyme isolated, ca. 0.3 mg of pure pol delta from 0.75 kg of calf thymus, is about 15-fold greater than can be achieved by conventional procedures. This procedure provides a significant advance in the isolation of pol delta in allowing its facile isolation from tissues in good yield. The isolated enzyme consisted of two subunits of 125 and 50 kDa. Characterization of the enzyme showed that these two subunits remained associated on glycerol gradient ultracentrifugation even in the presence of 2.8 M urea.

A transformed human hepatoma cell line was examined to determine if it was an appropriate model system for studying the mechanism of action of two peroxisome proliferators that lower blood lipids. Cultures of HepG2 cells were exposed to four different concentrations of either the hypolipidemic drug, clofibric acid (CLO), or the adrenal steroid, dehydroepiandrosterone (DHEA). Activities of two peroxisomal enzymes, palmitoyl-CoA oxidase and catalase, and two mitochondrial enzymes, carnitine palmitoyl-CoA transferase and succinate-INT-reductase, were measured in CLO- and DHEA-treated cells. In general, as the concentration of these hypolipidemic agents increased from 0 to 1000 microM, the specific activities of peroxisomal palmitoyl-CoA oxidase and catalase increased, and mitochondrial carnitine palmitoyl-CoA transferase and succinate-INT-reductase decreased. The activity of lactate dehydrogenase was significantly higher in the medium of cultures exposed to the 500 and 1000 microM concentration of DHEA compared with the control cultures, indicating the cytotoxic effects of this steroid at millimolar levels in vitro. In summary, the peroxisomal proliferators, DHEA and CLO, inversely altered peroxisomal and mitochondrial beta-oxidation in HepG2 cultures, but not to the extent reported for rat hepatocytes in vitro. In vitro concentrations of DHEA greater than 500 microM adversely affected the viability of HepG2 cells. The results of this study suggest that beta-oxidation in this human hepatoma cell line may not be as sensitive to hypolipidemic agents as are primary cultures of rat hepatocytes.

Genomic DNA from 32 unrelated families with male-limited precocious puberty was examined for the previously described Asp-578-->Gly, Met-571-->Ile, and Thr-577-->Ile mutations in transmembrane helix 6 of the human luteinizing hormone receptor (hLHR). Twenty-eight families had the inherited form of the disorder, and of these, 24 were found to have the Asp-578-->Gly mutation. Four additional mutations were found among the remaining four families with the inherited form and in four sporadic cases of the disorder: an A-->C transversion resulting in substitution of leucine for Ile-542 in the fifth transmembrane helix, an A-->G transition resulting in substitution of glycine for Asp-564 in the third cytoplasmic loop, a G-->T transversion resulting in substitution of tyrosine for Asp-578 in the sixth transmembrane helix, and a T-->C transition resulting in substitution of arginine for Cys-581 in the sixth transmembrane helix. Human embryonic kidney cells transfected with cDNAs for each of the mutant hLHRs, created by PCR-based mutagenesis of the wild-type hLHR cDNA, exhibited increased levels of basal cAMP production in the absence of agonist, indicating constitutive activation of the mutation hLHRs. Three of the additional mutations had specific features: Ile-542-->Leu and Cys-581-->Arg appeared ligand-unresponsive, whereas Asp-578-->Tyr appeared to correlate genotype with phenotype. We conclude that the region spanning nt 1624-1741 of exon 11 is a hotspot for heterogeneous point mutations that constitutively activate the hLHR and cause male-limited precocious puberty.

The pregnancy-specific glycoproteins (PSGs) of the placenta, members of the immunoglobulin superfamily, are encoded by multiple linked genes located on chromosome 19. To study the control of PSG expression, we have immortalized differentiated human placental cells (HP-A1) temperature-sensitive for transformation by a recombinant adenovirus-(ori-)-SV40 tsA mutant virus. We now show that expression of the PSG gene in HP-A1 cells is temperature-sensitive. At the permissive temperature (33 degrees C), these cells expressed low levels of PSG mRNA and synthesized a 64-kDa PSG. Shifting HP-A1 cells to a nonpermissive temperature (39.5 degrees C) increased PSG mRNA expression and biosynthesis with preferential increase in the synthesis of a 54-kDa and a low level of a 72-kDa PSG. Moreover, PSG expression was greatly induced by 5-bromo-2'-deoxyuridine (BudR), which selectively increased synthesis of PSGs of 72 and 54 kDa. In the presence of BudR, HP-A1 synthesized PSGs of 72, 64, and 54 kDa, similar to the pattern seen with placental PSGs. Ribonuclease protection assays demonstrated that HP-A1 cells express the majority of PSG mRNAs and BudR stimulated expression of PSG1 and PSG1-like transcripts. Reverse transcription and polymerase chain reaction analysis using PSG gene-specific primers demonstrated that untreated HP-A1 cells expressed primarily PSG1, PSG2, PSG4, and PSG5 mRNAs. BudR stimulated the expression of all PSG transcripts except PSG4. Moreover, in transient expression assays, BudR increased chloramphenicol acetyltransferase (CAT) expression directed by PSG1-I, PSG4, PSG5, PSG6, and PSG11 promoter-CAT fusion genes.(ABSTRACT TRUNCATED AT 250 WORDS)

A sensitive gas chromatographic method has been established for the simultaneous determination of biologically active inorganic anions, including cyanide, iodide, nitrite, sulfide, and thiocyanate anions as their volatile organic derivatives. The method is based on the formation of ion pairs from the anions and a complex cryptand and on the resulting neutral ion-pair partition to an organic phase for derivatization with pentafluorobenzyl bromide. Several parameters affecting the partition and derivatization of the anions were investigated. Individual and simultaneous determination of the anions can be achieved at sub-nmol levels with an electron-capture detector. Partial application of the method for the analysis of cyanide, nitrite, and thiocyanate in real samples proved satisfactory.

Effects of extracellular calcium on signal transmission between photoreceptors and horizontal cells (HCs) are studied in superfused isolated retina of the larval tiger salamander. Horizontal cell light response is optimal when extracellular Ca2+ is maintained between 1-2 mM. Ca2+ levels beyond this range in either direction significantly reduce the HC light response amplitude. When extracellular Ca2+ is lowered from 2 mM to 0.5 mM, the rod input to HCs is reduced whereas the cone input is not affected. In comparison, the peak voltage responses of rods are not changed whereas the cone voltage responses are enhanced in 0.5 mM Ca2+. The selective suppression of rod input to HCs is probably due to the interplay of three factors: (1) the photocurrents, (2) voltage- and time-dependent membrane currents in photoreceptors, and (3) the Ca(2+)-dependent synaptic gain between photoreceptors and HCs.

Percutaneous subclavian implantation of an indwelling central venous catheter is an easy technique and provides convenient venous access for long-term intravenous therapy. Although rarely reported, spontaneous fracture of the catheter is an ominous complication which requires a prompt diagnosis and urgent treatment. We present a case of "pinch-off sign" resulting in a spontaneous fracture of an indwelling central venous catheter. A 49-year-old female breast cancer patient was admitted and Port-A-Cath was implanted for chemotherapy. Immediately after the implantation, fluid infusion and blood withdrawal was smooth until clinical "pinch-off sign" developed 3 weeks later. Chest X ray revealed no abnormal findings. Extravasation of antineoplastic drugs was noted 113 days after operation. Fracture of the indwelling catheter was found at the clavicle-rib junction. The fractured fragment was removed with a transvenous snare under fluoroscope. There was no hemodynamic derangement during the peri-operative period. Microscopy studies suggested that intermittent pressure on the catheter between the clavicle and the first rib may be responsible. The catheter wore on the medial side ue to a tearing and scissoring effect associated with free shoulder joint movement exerted additional forces on this wearing point which led to catheter fracture. The relationship between the spontaneous catheter fracture and "pinch-off sign" is reviewed. Our suggestions are: (1) By avoiding the traditional cannulation of the median subclavian vein, the lateral subclavian vein, infraclavicular axillary vein or internal jugular vein should be better routes for implantation. (2) Chest X ray (anterior-posterior and lateral view) should be examined routinely 3 weeks after the operation.(ABSTRACT TRUNCATED AT 250 WORDS)

Background: Compensation inequity by gender is a problem across occupations in the United States. Most compensation-monitoring efforts in academic medicine have been informal. The authors developed an analytic method for formal, ongoing evaluation of compensation equity in academic medicine., Method: A historical cohort study was conducted at Michigan State University College of Medicine using data from 1990, 1991, and 1992 to (1) evaluate methods for monitoring compensation equity, (2) test the feasibility of compensation-equity monitoring as part of administrative information systems, and (3) determine whether compensation inequity existed in a case study of faculty salaries. Internal market adjustments for specialty, clinical or basic science "type," and calendar- or academic-year appointments were made before establishing a male cohort for each female faculty member., Results: The method developed appears feasible for routine administrative monitoring of compensation equity. When the compensations of women of each type and rank were compared with the compensations of their male cohorts, inequities appeared to exist for basic scientists, but not clinicians, based on a criterion of the groups' compensations being 4% or more below those of their cohorts for two successive years., Conclusion: The authors suggest that formal monitoring of compensation equity is an important and feasible administrative undertaking to correct historical inequities. This is an area in which leadership by U.S. medical colleges is needed.

Zinc ions are colocalized with glutamatergic synaptic vesicles in vertebrate photoreceptors and may act as a diffusible molecular switch regulating neurotransmitter signaling at two distinct sites in the outer retina. In the dark, extracellular zinc acts presynaptically at rods and cones to minimize the depletion of tonically released glutamate, and selectively reduces GABA-mediated depolarization in horizontal cells, accelerating the response kinetics of the second-order cells. The discovery of zinc ions in photoreceptors provides a mechanism for gain control, kinetics modulation, and the balance of rod vs cone output at the first synapse in the visual system.

The total cortisol content in tilapia was 55 pg immediately following fertilization, then decreased abruptly and maintained a lower level of 10-20 pg until hatching; after hatching the cortisol content increased to 50 pg by the 7th day. Fertilized eggs were incubated in either 32% saltwater or fresh water and sampled at various developmental stages. Both groups showed dramatic changes in cortisol content following development. However, no significant difference in the cortisol level was found between the two groups. Tilapia larvae, hatched in fresh water, were reared with feed containing 0 (control) or 150 mg/kg wt/day cortisol, corticosterone, 17 alpha-hydroxyprogesterone (17 alpha-OHP) or 11 alpha-deoxycortisol (11 alpha-DC) for 8 days and then transferred directly to 27.5% saltwater. Those reared with corticosterone, 17 alpha-OHP, or 11 alpha-DC, similar to the control, all died within 4-8 hr after the transfer. However, the larvae treated with cortisol showed a much higher survival rate of 40-60%. The tissue osmolality in the control larvae, 394.3 +/- 3.7 (mmol/kg), increased abruptly after transfer to 26% saltwater and reach a peak, 681.5 +/- 47.5, before the larvae all died (12th hr after the transfer). In contrast, tissue osmolality in cortisol-treated (150 mg/kg wt/day for 12 days) larvae was 570.7 +/- 62.6 at the 12th hr and then began to decrease to 448.5 +/- 9.4 at the 24th hr and 386.0 at the 48th hr. These findings suggest that cortisol could play a critical role in the hypoosmoregulation in tilapia larvae.

Application of gamma-aminobutyric acid (GABA) slows down the horizontal cell response time course (HCRRT) and induces membrane depolarization in horizontal cells (HCs) after synaptic inputs are blocked by Co2+. We present evidence that suggests both effects are probably mediated by GABAA receptors which open chloride channels in the HC membrane. In any given concentration of GABA, ranged from 0 to 100 microM, the HC membrane potential (VHC) in saturating light and in the presence of 100 microM Co2+ are identical. This result suggests that GABA in both light and 100 microM Co2+ opens the same amount of chloride channels (same gCl) so that VHC determined by chloride and leak conductances has the same value. Higher concentrations of Co2+ (> 300 microM) not only blocks synaptic transmission from photoreceptors to HCs, but also acts as an antagonist that suppresses the GABA-mediated depolarization in HCs.

Synaptic transmission between photoreceptors and bipolar cells was studied in dark-adapted tiger salamander retinas. Based on the relative light sensitivity, bipolar cells, either depolarizing (DBC) or hyperpolarizing (HBC), fell into two groups: one receives inputs primarily from rods (rod-dominated bipolar cells, DBCR and HBCR) and the other receives inputs primarily from cones (cone-dominated bipolar cells, DBCC and HBCC). The input-output relations of the rod-DBCR and rod-HBCR synapses were determined by plotting the voltage responses of the rod and DBCR (or HBCR) to dim 500-nm light steps, which polarizes only the rods but not the cones. The slope gains of both synapses were the highest near the dark rod voltage (-2.5 for the rod-DBCR synapse and 4.0 for the rod-HBCR synapse), and they (the absolute values) became progressively smaller at more hyperpolarized rod voltages.

1. The effects of glutamate receptor agonists and antagonists on bipolar cells and ganglion cells were studied with the use of intracellular and extracellular recording in the superfused, isolated, flat-mounted tiger salamander retina. The goal of the experiments was to correlate glutamate receptor subtypes with their localization at specific synaptic sites in the tiger salamander retina. The drugs tested were the kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the N-methyl-D-aspartate (NMDA) receptor antagonist 3-(C+/-)-2-carboxy-piperazin-4-yl)-propyl-1-phosphonic acid (CPP) and L-2-amino-4-phosphonobutyrate (L-AP4). 2. The light responses of hyperpolarizing bipolar cells were suppressed by 20 microM CNQX, whereas L-AP4 had no effect on their light responses. In contrast, 20 microM CNQX had no effect on depolarizing bipolar cells, whereas L-AP4 abolished the light responses of these cells. 3. The light offset responses of OFF and ON-OFF ganglion cells were completely blocked by concentrations of CNQX as low as 5 microM. The light onset responses of ON-OFF ganglion cells were blocked when the concentration of CNQX was raised to 20 microM. In addition, 30 microM CPP partially blocked the light onset responses of ON-OFF ganglion cells but had a lesser effect on the light offset responses. 4. Twenty micromolars of CNQX blocked a transient component, and 20 microM CPP blocked a sustained component of the light response of sustained-ON ganglion cells.(ABSTRACT TRUNCATED AT 250 WORDS)

1. The relative contribution of rod and cone inputs to bipolar and ganglion cells were studied by comparing the response-irradiance relations, spectral sensitivities, and response waveforms of these neurons recorded from the isolated, flat-mounted tiger salamander retina under dark-adapted conditions. 2. Bipolar cells could be differentiated both on the basis of the polarity of the light response and on their relative rod/cone input. Thus some depolarizing bipolar cells appeared more strongly influenced by rod input (DBCR), whereas others were more influenced by cone input (DBCC). Similarly, hyperpolarizing bipolar cells could be divided into those that received rod-dominant input (HBCR) or cone-dominant input (HBCC). 3. The light onset response of sustained-ON ganglion cells reflected both rod-dominant input from DBCRs and cone-dominant input from DBCCs. 4. OFF ganglion cells displayed both a rod-dominant sustained light offset response and a cone-dominant transient light offset response, suggesting input from both HBCRs and HBCCs. 5. In ON-OFF ganglion cells, the light onset response was strongly rod dominated and was presumably mediated by DBCRs, whereas the light offset response displayed both rod and cone influence, suggesting input from HBCRs and HBCCs. The contribution of cones to the light onset response of ON-OFF ganglion cells was only observed in the presence of a rod-adapting background light. 6. A suppression of the light offset responses of OFF and ON-OFF ganglion cells was observed, which was dependent both on the wavelength and irradiance of the light stimulus. 7. These results indicate that the photoreceptor inputs to bipolar cells in the tiger salamander retina are segregated such that they form separate rod-dominant and cone-dominant pathways. Thus the response properties of the different types of ganglion cells are influenced not only by the excitatory and inhibitory inputs they receive from the bipolar and amacrine cells but also whether these inputs are provided through rod-dominant or cone-dominant pathways. The functional implications of these findings are discussed.

The presence of PSG in blood cells has been demonstrated by immunohistochemical staining. However, the origin of those proteins is not known. This report examines the expression of the PSG genes in different types of freshly isolated blood cells. RNA isolated from bone marrow and peripheral blood cells of healthy individuals was analyzed for PSG transcripts by reverse transcriptase-polymerase chain reaction using synthetic oligonucleotide primers specific for the PSG genes. The level of expression of the PSG genes in different types of cells exhibited significant individual variation. Trace amounts of PSG transcripts could be detected in polymorphonuclear cells (PMN), monocytes and B lymphocytes while T lymphocytes always contained the highest level of transcript. The expression of PSG genes in the blood cells apparently was not affected by the method of isolation nor by overnight culturing of these cells except in the case when lymphocytes were separated by rosetting with sheep red blood cells. All reported PSG transcripts were detected in blood cells. Both type I and type II transcripts of the PSG genes were detected in blood cells with the exception of type II transcript of PSG5 and PSG11 which were only found in the placenta. Tissue specificity in the expression or alternative splicing of some of the PSG family members was implicated.

Dietary, anthropometric, and chronic disease risk factors (CDRF) including blood lipids and blood pressure (BP), were measured in 91 vitamin-mineral supplement users (SU) and nonusers (NU) representing a wide range of athletic interests. Supplements were used by 46 (51%) subjects; 100% of female athletes and 51% of male athletes used supplements while none of a group of 15 control female subjects currently used supplements. Both dietary intake and energy expenditure were measured using 7-day records. Adiposity was determined from body weight, body mass index, and skinfolds. Total cholesterol, high-density lipoprotein cholesterol, serum ferritin, hemoglobin, hematocrit, zinc, copper, and vitamin C were based on 12-hour fasting blood samples. Dietary intake (excluding supplements) for SU tended to be greater than NU for vitamin C, thiamin, riboflavin, niacin, B6, B12, folate, calcium, iron and magnesium. Plasma vitamin C levels were significantly higher among SU than NU of both gender groups (p < 0.05). Although SU may exhibit additional healthy lifestyle practices, lipid profiles for many of these athletes were unfavorable with regard to CDRF.

Although synaptic transmission in the retina has been assumed to be static, it appears that the voltage gains of the synapses between photoreceptors and second-order cells can be enhanced by light. Voltage gains of the synapses between rods and bipolar (or horizontal) cells are about 10 times higher in the presence of dim background light than in darkness. This increase in synaptic gain may compensate for the loss of rod light responsiveness caused by weak background light so that the animal can maintain good rod sensitivity under moonlight or starlight, the natural lighting condition for mating and food catching.

The purpose of this research was to measure changes in selected plasma amino acids (AA) during two successive exercise trials to exhaustion. Eleven trained male athletes completed these trials at weeks 4, 6, 8 and 12. Blood samples for each test were collected after a 12-hour fast at times (in minutes) 0 (Resting), 45, 90, 135, 180, at exhaustion (EI), after a 20-minute recovery period, and at the second exhaustion (EII). At the end of EI, subjects consumed an artificially sweetened water replacement (placebo) treatment or a carbohydrate (CHO) replacement (1.1 g CHO/kg BW) in order to determine any effect of CHO replacement on changes in energy substrates or AA, adjusted for plasma volume changes. From baseline to EI, alpha-aminobutyric acid, alanine, glycine, isoleucine, serine, valine threonine, and tyrosine decreased significantly (p less than or equal to 0.05), while taurine increased significantly. During the recovery period following EI, isoleucine, leucine, ornithine, phenylalanine, tyrosine, urea and valine increased significantly. From the end of recovery until EII, alanine, aspartic acid, glycine, isoleucine, leucine, ornithine, phenylalanine, serine, threonine, tyrosine and valine decreased significantly. CHO replacement had no effect on the mean change scores for any AA from EI to the end of the recovery period and affected only serine, citrulline, glycine and threonine from the end of the recovery period to EII.

The primary feedback control apparatus in the outer retina is the sign-inverting feedback synapse between horizontal cells and cones. In many lower vertebrates horizontal cells release GABA in darkness, which opens Cl- channels in cones. Input-output relations of the feedback synapse reveal that the synaptic gain is light-dependent with the highest negative gain near the dark horizontal cell potential. The horizontal cell-cone feedback synapse improves the reliability of the photoreceptor output synapses. It also modulates the dynamic range and mediates color opponency and surround responses in second-order retinal neurons.

The whole-animal content of the cortisol was measured in embryos and larvae of tilapia (Oreochromis mossambicus), rainbow trout (Oncorhynchus mykiss), ayu (Plecoglossus altivelis), milkfish (Chanos chanos), and yellowfin bream (Acanthropagrus latus) by radioimmunoassay following the validation of an extraction method. The total cortisol content in tilapia was 50.3 +/- 19.1 pg immediately following fertilization, then decreased abruptly and maintained a lower level of 10-17 pg until larval hatching; after hatching the cortisol content increased to 47.2 +/- 11.9 pg by the seventh day. Newly hatched rainbow trout had 60.3 +/- 6.4 pg cortisol and then increased their cortisol level slowly to 83.0 +/- 7.2 pg by the fifth day after hatching. Ayu larvae contained 5.2 pg cortisol immediately following hatching. On the other hand, pelagic milkfish revealed a much lower cortisol level, being undetectable from hatching until the second day and ranging from 0.4 to 3.7 pg from the third to seventh day after hatching. Yellowfin bream, demonstrating a similarity to milkfish, were not found to have any detectable cortisol from hatching until the third day, but presented 1.6-7.7 pg from the fifth to seventh day after hatching. The presence and clearance of cortisol during early development of fertilized eggs of tilapia suggest a maternal origin of the hormone. The amount of cortisol deposited in the larval body of tilapia increased after hatching from 25% to nearly 100% of the total cortisol in whole larvae, while that in the larval yolk sac decreased to an undetectable level, implying that the increased cortisol may be produced or secreted by the larva. The possible role of cortisol in larval development is discussed.

A simple and economical high-performance liquid chromatographic method has been developed for the simultaneous determination of betamethasone and dexamethasone. The method is based on the derivatization of the structural epimers of betamethasone and dexamethasone with a homochiral reagent, N-carbobenzoxy-L-phenylalanine. The derivatives obtained were easily recognized by a non-chiral silica column with n-hexane-dichloromethane-isopropanol (100:100:4, v/v/v) as a mobile phase and a good separation was obtained for quantitation. The method was satisfactorily applied to the determination of betamethasone and dexamethasone in tablets.

The purpose of this study was to examine the relationships of several cardiovascular disease risk factors [blood pressure (BP), total cholesterol (TC), high-density-lipoprotein cholesterol (HDL-C), and the HDL-C:TC ratio], as well as the body mass index (BMI) and percent body fat with daily energy intake (EI) and daily energy expenditure (EE) of North Carolina farmers and their wives. Data were collected from 195 subjects. Daily EI and EE were estimated from 4-day food and 4-day activity records, respectively, collected on the same days. Pearson correlation coefficients for chronic disease risk factors with both EI and EE were generally low. When compared to EI, EE was more highly correlated with both lean body mass (r = 0.88) and BMI (r = 0.73), and was less time consuming and easier for the subjects to use. EE obtained from a reliable activity record may be a more practical tool for assessing the possible relationship(s) of energy metabolism to chronic disease risk factors.

The cDNA for human gamma-glutamyl carboxylase, which accomplishes the post-translational modification required for the activity of all of the vitamin K-dependent proteins, was cloned. The enzyme is a 758-residue integral membrane protein and appears to have three transmembrane domains near its amino terminus. The hydrophilic COOH-terminal half of the carboxylase has 19.3 percent identity with soybean seed lipoxygenase. Expression of the cloned cDNA resulted in an increase in carboxylase activity in microsomes of transfected cells compared to mock-transfected cells.

An adsorption thin-layer chromatography (TLC) is described for the detection of glucocorticoids including triamcinolone, prednisolone, 6 alpha-methylprednisolone, betamethasone, dexamethasone, hydrocortisone, prednisone and cortisone. Separation of the glucocorticoids was performed on a silica stationary phase with chloroform-dioxane-methanol (15:4:1, v/v) as a mobile phase. The complete resolution of the epimers of betamethasone and dexamethasone was attained by derivatization of them with (1S)-(-)-camphanic chloride using triethylamine as a catalyst. The application of the method to the analysis of glucocorticoids in various samples is being smoothly developed.

Effects of the major glutamate receptor agonists, kainate (KA), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), quisqualate (QA), N-methyl-D-aspartate (NMDA), L-alpha-amino-4-phosphonobutyrate (L-AP4), and trans-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) on horizontal cells (HCs) were studied in superfused larval tiger salamander retina. 20 microM of KA, AMPA, and QA mimicked the action of 3 mM glutamate in the absence and presence of 1 mM Co2+. 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) blocked the actions of KA and AMPA, but not those of QA and glutamate, indicative of the existence of CNQX-resistant QA receptors in the tiger salamander HCs. Prolonged application of ACPD hyperpolarized the HCs and enhanced the light responses, probably by shifting the resting HC voltage (Er) to a more hyperpolarized position. It is possible that the KA, AMPA, and CNQX-resistant QA receptors are involved in mediating the postsynaptic light responses in HCs, and ACPD receptors are involved in sensitivity adjustment of the HC responses.

Dietary, anthropometric, and chronic disease risk factors (CDRF) in vitamin/mineral supplement users (U) and non-users (NU) were measured in a farm population consisting of 162 subjects (46% females and 54% males; 20-79 years of age, mean age: 52 years). Subjects were white, except for two black males. Supplements were used by 62 subjects (38%); 47% of females and 31% of males used supplements; 43% of subjects over 50 years of age and 32% of subjects age 50 or under used supplements. Both dietary intake and energy expenditure were measured using 4-day records. Indices of adiposity included body weight, BMI, and estimated body fat. Total cholesterol (TC), high-density-lipoprotein cholesterol (HDL-C), serum ferritin, hemoglobin, hematocrit, zinc, copper, and vitamin C were based on 12-hr fasting blood samples. Dietary intake (excluding supplements) for vitamin/mineral U was greater than NU for vitamin C (p = 0.006), thiamin (p = 0.01), riboflavin (p = 0.03), niacin (p = 0.02), folic acid (p = 0.001), vitamin B6 (p = 0.01), and magnesium (p = 0.019). Vitamin C levels were significantly higher and the sum of four skinfolds was significantly lower among U than NU. In this population, 24% of males and 18% of females had TC levels over 240 mg%; 8% of both males and females had blood pressures (BP) greater than 140/90 mm Hg, while 49% of males and 46% of females had BP between 120/80 and 140/90; and 71% of males were more than 25% fat, and 56% of females were more than 35% fat. Despite the high prevalence of CDRF, there were no significant differences between supplement U and NU.

1. The input-output relation of the feedback synapse between horizontal cells (HCs) and cones was studied by simultaneously recording the light responses of the HCs and of cones the outer segments of which were truncated off. 2. The postsynaptic light response of the truncated cone was depolarizing and free of direct influence of photocurrents. These postsynaptic light responses were graded and sustained; their waveform resembled that of the HC light responses. 3. Input-output relation of the HC-cone feedback synapse was obtained by plotting the simultaneous voltage points of the HC and truncated cone light responses. At the resting potential of the cone (-40 mV), the voltage gain of the feedback synapse was about -0.33 when VHC = -20 mV and it was about -0.03 when VHC = -60 mV. 4. At more hyperpolarized cone voltages, the feedback signals in cones became smaller, and they reversed at about -67 mV. The voltage gain of the feedback synapse at VHC = -20 mV was about -0.23, -0.18, -0.07, and +0.2 when Vcone = -44.5, -52.5, -65, and -77.5 mV, respectively. 5. Light hyperpolarized the HC, which resulted in a conductance change (delta Gs) in cones. The cone conductance decreased progressively as the HCs were increasingly hyperpolarized, and delta Gs reached a maximum value of 0.93 nS when the HCs were hyperpolarized from -20 to -52 mV. 6. The peak light responses of intact cones were plotted against the peak HC light responses. This gives the relationship between the pre- and postsynaptic voltages of the cone-HC (forward) and HC-cone (feedback) synapses at any given light intensity. Combining this relationship with the input-output relations obtained at various voltages of the truncated cones allows the input-output relation of the feedback synapse for light-evoked signals to be obtained. 7. The input-output relation of the feedback synapse for light-evoked signals was bell-shaped, because the feedback light responses were controlled by two opposing factors: as the light became brighter, the postsynaptic conductance change increased, but the driving force decreased. 8. For light-evoked signals, the slope gain (slope of the input-output relation) of the HC-cone feedback synapse was negative (varied from -0.33 to 0) when VHC lay between -20 and -52 mV; and it was positive (0 to +0.8) when VHC lay between -52 and -72 mV. 9.(ABSTRACT TRUNCATED AT 400 WORDS)

Lateral inhibition is the ubiquitous strategy used by visual neurons for spatial resolution throughout the animal kingdom. It has been a puzzle whether lateral inputs in retinal bipolar cells are mediated by the horizontal cell (HC)-cone feedback synapse, by the HC-bipolar cell feedforward synapse, or by both. By blocking the central inputs of the depolarizing bipolar cells (DBCs) with L-2-amino-4-phosphonobutyrate, we were able to eliminate the contribution of the feedback synapse and to demonstrate the postsynaptic light response in DBCs mediated by the HC-DBC feedforward synapse. The HC-DBC feedforward synapse contributes roughly one-third of the surround response whereas the HC-cone-DBC feedback synapse probably contributes the rest.

Nine eumenorrheic women were studied at five separate times each month for a minimum of 2 months. Fasting bloods and 1-day food records were obtained from all women at these times to describe the pattern and magnitude of within-person variation in selected nutrient and lipid indicators attributable to menstrual cyclicity. Serum cholesterol, beta-carotene, 17 beta-estradiol (E2), and dietary selenium intakes exhibited significant periodic regressions against time. For the group as a whole, however, only serum cholesterol and E2 measures exhibited significantly strong consistent periodicities. Future studies with a larger sample of women are warranted to confirm these findings. Unless strict blood sampling protocols are followed, variation attributable to menstrual cyclicity may mitigate the reliability of the serum cholesterol screenings advocated in the past few years.

Vitamin K-dependent carboxylase catalyzes the modification of specific glutamic acids to gamma-carboxyglutamic acid in several blood-coagulation proteins. This modification is required for the blood-clotting activity of these proteins and has thus been the subject of intense investigation. We have now identified the bovine vitamin K-dependent carboxylase and purified it to near homogeneity by an affinity procedure that uses the 59-amino acid peptide FIXQ/S (residues -18 to 41 of factor IX with mutations Arg----Gln at residue -4 and Arg----Ser at residue -1). The carboxylase as purified has a molecular weight of 94,000. It is also the major protein that can be cross-linked to iodinated FIXQ/S and is the only protein whose cross-linking is prevented by a synthetic factor IX propeptide. The degree of purification is about 7000-fold with reference to ammonium sulfate-fractionated microsomal protein from liver.

1. The influence of rod adaptation on cone-mediated intracellular responses of distal retinal neurons was examined in the larval tiger salamander. Rods were selectively stimulated by the use of 450-520 nm adapting stimuli too dim to appreciably influence cones. Cones were primarily stimulated with the use of deep-red stimuli (maximally sensitive to wavelengths greater than 650 nm). The qualitative properties of rod-cone interaction were assessed with the use of several different photic-stimulus paradigms. 2. Confirming results of prior studies, we showed that rod adaptation changed the time course of cone-mediated responses to the onset of square-wave light flashes in horizontal cells (HCs); rod adaptation had no similar influence in other distal retinal neurons. Rod adaptation also enhanced cone-mediated responses to rapid flicker in cones, hyperpolarizing (HPBCs) and depolarizing (DPBCs) bipolar cells, as well as HCs. 3. We also examined the influence of rod-stimulating background fields on cone-mediated responses to slow (approximately 1-Hz) flicker composed of sawteeth with a rapid onset (ramp offset) or with a rapid offset (ramp onset). Such stimulation maintained a constant state of long-term adaptation while providing cones with transient-ON or transient-OFF stimulation. 4. Rod adaptation speeds up the response of HCs to rapid onset and increases response amplitude. Rod adaptation had no reliable influence on response to rapid onset in cones or bipolar cells. 5. Rod adaptation enhanced the amplitude of responses of HCs to rapid offset without altering response time course. 6. Rod adaptation greatly enhanced the amplitude of DPBC responses to rapid offset having no reliable influence on the time course of the response. 7. Rod adaptation caused responses of HPBCs to rapid offset to become much more transient. Rod backgrounds had a similar but smaller and less reliable influence on the response of cones to rapid offset. 8. The foregoing results indicate that rod adaptation exerts a minimum of two separate influences on cone-mediated responses in distal amphibian retina. Changes at light onset must reflect the operation of a mechanism that alters response kinetics of the HC membrane. Changes at light offset reflect the operation of a separate mechanism or set of mechanisms that must act in part presynaptically to the HCs.

We report the expression in Escherichia coli of a fusion protein that contains the propeptide sequence and gamma-carboxyglutamic acid domain (residues -18 to 41) of human factor IX (FIXGla). CNBr was used to release FIXGla from the fusion protein. The 59-amino acid peptide is an efficient substrate for in vitro gamma-carboxylation. Its Km,app (0.55 microM) is several thousand-fold lower than that of the commonly used substrate FLEEL and about 5 times lower than proPT28 or proFIX28, (Hubbard, B. R., Jacobs, M., Ulrich, M. M. W., Walsh, C., Furie, B., and Furie, B. C. (1989) J. Biol. Chem. 264, 14145-14150). In addition, FIXGla is the first peptide substrate that is carboxylated in vitro to more than one gamma-carboxyglutamic acid/molecule (6-11 gamma-carboxyglutamic acids/molecule). We created peptides with mutations identical to FIXSan Dimas or FIXCambridge as well as a peptide with both mutations in the propeptide sequence and examined the effect of the mutations on in vitro carboxylation. Enzyme kinetic studies revealed no significant difference in Vmax/Km values between normal and mutant substrates. Maximum carbon dioxide incorporation was achieved with the double mutant. From these data we conclude the following. 1) FIXGla and its mutants are excellent substrates for studying the mechanism of gamma-carboxylase. 2) Although arginines at positions -4 and -1 are highly conserved in the propeptide sequence of all the vitamin K-dependent proteins, neither is critical for gamma-carboxylation.