C/EBP alpha:AP-1 leucine zipper heterodimers bind novel DNA elements, activate the PU.1 promoter and direct monocyte lineage commitment more potently than C/EBP alpha homodimers or AP-1.

The basic-region leucine zipper (BR-LZ or bZIP) transcription factors dimerize via their LZ domains to position the adjacent BRs for DNA binding. Members of the C/EBP, AP-1 and CREB/ATF bZIP subfamilies form homodimeric or heterodimeric complexes with other members of the same subset and bind-specific DNA motifs. Here we demonstrate that C/EBPalpha also zippers with AP-1 proteins and that this interaction allows contact with novel DNA elements and induction of monocyte lineage commitment in myeloid progenitors. A leucine zipper swap:gel shift assay demonstrates that C/EBPalpha zippers with c-Jun, JunB or c-Fos, but not with c-Maf or MafB. To evaluate activities of specific homodimers or heterodimers we utilized LZs with acid (LZE) or basic (LZK) residues in their salt bridge positions. C/EBPalphaLZE:C/EBPalphaLZK preferentially binds a C/EBP site, c-JunLZE:c-FosLZK an AP-1 site and C/EBPalphaLZE:c-JunLZK a hybrid element identified as TTGCGTCAT by oligonucleotide selection. In murine myeloid progenitors, C/EBPalpha:c-Jun or C/EBPalpha:c-Fos LZE:LZK heterodimers induce monocyte lineage commitment with markedly increased potency compared with C/EBPalpha or c-Jun homodimers or c-Jun:c-Fos heterodimers, demonstrating a positive functional consequence of C/EBP:AP-1 bZIP subfamily interaction. C/EBPalpha:cJun binds and activates the endogenous PU.1 promoter, providing one mechanism for induction of monopoiesis by this complex.

Gastrocnemius and soleus muscle length, velocity, and EMG responses to changes in pedalling cadence.

Several authors have shown different excitation patterns for soleus and gastrocnemius muscles in response to cadence manipulation during cycling. The purpose of this study was to examine gastrocnemius and soleus length and velocity change as a function of pedalling cadence to consider mechanisms underlying these excitation differences. Ten male and two female cyclists rode at five randomly assigned cadences (50, 65, 80, 95, and 110 rpm) at a nominal 200 W power output while EMG of the gastrocnemius and soleus and sagittal plane video were recorded. Joint-coordinate data for the knee and ankle were used with equations of Grieve et al. [Grieve D, Pheasant S, Cavanagh PR. Prediction of gastrocnemius length from knee and ankle joint posture, in: E. Asmussen, K. Jorgensen, editors. International Series on Biomechanics, vol. 2A, Baltimore: University Park Press; 1978. p. 405-412] to compute gastrocnemius and soleus length and velocity. Consistent with previous publications, gastrocnemius displayed a significant (p<0.05) increase in integrated EMG with increased cadence, whereas cadence had no significant effect on integrated EMG of the soleus. The ankle became significantly (p<0.05) more plantar flexed and reflected a reduced range of motion with increased cadence while the knee became significantly (p<0.05) less extended. Soleus decreased its range of motion by 29%, whereas gastrocnemius decreased its range of motion by 9%. In contrast, soleus increased its velocity range by 32% and gastrocnemius increased by 45%. These data show that with increased cadence gastrocnemius operated over a narrower range of operating lengths but at a higher range of shortening velocity than soleus. The higher range of velocity may have resulted in the need for a relatively higher excitation, as indicated by the integrated EMG, as the muscle was working at a different range on its force-velocity curve. During the recovery portion of the pedalling cycle, the soleus was acting eccentrically while the gastrocnemius acted concentrically indicating the triceps surae complex did not always act in unison.

Targeting of G protein-coupled receptors to the basolateral surface of polarized renal epithelial cells involves multiple, non-contiguous structural signals.

Truncations and chimeras of the alpha2A-adrenergic receptor (alpha2AAR) were evaluated to identify membrane domains responsible for its direct basolateral targeting in Madin-Darby canine kidney cells. An alpha2AAR truncation, encoding transmembrane (TM) regions 1-5, was first delivered basolaterally, but within minutes appeared apically, and at steady-state was primarily lateral in its immunocytochemical localization. A TM 1-5 truncation with the third intracellular loop revealed more intense lateral localization than for the TM 1-5 structure, consistent with the role of the third intracellular loop in alpha2AAR stabilization. Addition of TM 6-7 of A1 adenosine receptor (A1AdoR) to alpha2AARTM1-5 creates a chimera, alpha2AARTM1-5/A1AdoRTM6-7, which was first delivered apically, resulting either from loss of alpha2AAR sorting information in TM 6-7 or acquisition of apical trafficking signals within A1AdoRTM6-7. Evidence that alpha2AARTM6-7 imparts basolateral targeting information is revealed by the significant basolateral localization of the A1AdoRTM1-5/alpha2AARTM6-7 and A1AdoRTM1-5/alpha2AARTM6-7+i3 chimeras, in contrast to the dominant apical localization of A1AdoR. These results reveal that sequences within TM 1-5 and within TM 6-7 of the alpha2AAR confer basolateral targeting, providing the first evidence that alpha2AAR basolateral localization is not conferred by a single region but by non-contiguous membrane-embedded or proximal sequences.

Differential targeting and retention of G protein-coupled receptors in polarized epithelial cells.

Localization of receptors in discrete cellular microdomains undoubtedly contributes to their interaction with particular effectors and receptor targets. For G protein-coupled receptors, virtually nothing is known about the mechanisms and structural features responsible for their targeting to and retention in varying surface domains. We have shown that the Gi/ Go-coupled alpha 2A-adrenergic receptor (alpha 2AAR) is directly targeted to the lateral subdomain of MDCK II cells. Mutational analysis has revealed that regions in or near the bilayer are likely critical for alpha 2AAR targeting, whereas endofacial domains contribute to alpha 2AAR retention on the lateral surface. Although the alpha 2BAR also is enriched on the lateral subdomain at steady-state, its polarization occurs after initial random delivery to both apical and basolateral surfaces followed by a selective accumulation on the lateral subdomain. The alpha 2CAR also is expressed on the lateral subdomain and achieves its localization via direct delivery to the basolateral surface; however, the alpha 2CAR also exists in an as yet not fully characterized intracellular compartment. Interestingly, another Gi/Go-coupled receptor, the A1 adenosine receptor, is enriched on the apical surface of MDCK II calls and achieves this localization by direct apical delivery. These findings indicate that receptor delivery to polarized surfaces is not determined by receptor coupling to a specific subpopulation of G proteins.

Receptors coupled to pertussis toxin-sensitive G-proteins traffic to opposite surfaces in Madin-Darby canine kidney cells. A1 adenosine receptors achieve apical and alpha 2A adrenergic receptors achieve basolateral localization.

The alpha 2A adrenergic receptor (alpha 2AAR) previously was shown to be directly delivered to and retained on the lateral subdomain of renal epithelial cells. The present studies demonstrate that, in contrast, wild-type and epitope-tagged canine A1 adenosine receptors (A1AdoR) are apically enriched (65-83%) in Madin-Darby canine kidney (MDCKII) and porcine renal epithelial (LLC-PKI) cells, based on surface biotinylation strategies detecting photoaffinity-labeled A1AdoR. Confocal microscopy corroborated the apical enrichment of the epitopetagged A1AdoR. Metabolic labeling studies revealed that this steady-state polarization is achieved by direct delivery to both the apical (60-75%) and basolateral surface. Growth of A1AdoR-expressing cells as monolayers presence of A1AdoR antagonists, which decreased cell growth, suggesting that A1AdoR elicit MDCKII cell proliferation. The preferential apical but detectable basolateral localization of A1AdoR provides a molecular understanding of published reports that functional responses can be elicited following apical as well as basolateral delivery of adenosine agonists in varying renal preparations. These findings also suggest that receptor chimeras derived from the Gi/Go-protein-coupled alpha 2AAR and A1AdoR will be informative in revealing structural features critical for basolateral versus apical targeting.

Introduction of purified alpha 2A-adrenergic receptors into uniformly oriented, unilamellar, phospholipid vesicles: productive coupling to G proteins but lack of receptor-dependent ion transport.

Introduction of highly purified alpha 2A-adrenergic receptors (alpha 2AAR) into lipid vesicles resulted in vesicle preparations that were unilamellar in structure, nonleaky to monovalent cations, and uniformly oriented such that the cytoplasmic domains of the alpha 2AAR faced the vesicle exterior. In this orientation, addition of Gi/G(o) G proteins yielded a 4-5-fold stimulation of agonist-dependent guanosine-5'-O-(3-[35S]thio)triphosphate binding to the G protein alpha subunit. These nonleaky, uniformly oriented, alpha 2AAR-containing vesicle preparations allowed us to explore the hypothesis that the alpha 2AAR itself, or in combination with Gi/G(o) proteins, is able to effect ion translocation. Measurements of 22Na+ uptake, 22Na+ efflux, and H+ movement revealed no detectable agonist-stimulated, receptor-dependent, ion translocation, even in the presence of G proteins, suggesting that allosteric regulation of alpha 2AAR by cations and amiloride analogs is not an indication that the alpha 2AAR itself is an ion transporter. Nonetheless, the methodology developed in the present studies for preparation of nonleaky vesicles containing receptor and G proteins should be well suited for evaluating the stoichiometry and selectivity of receptor-G protein interactions and, in particular, G protein specificity in mediating receptor-dependent regulation of voltage-gated or receptor-operated ion channels.

Unique structural features important for stabilization versus polarization of the alpha 2A-adrenergic receptor on the basolateral membrane of Madin-Darby canine kidney cells.

The alpha 2A-adrenergic receptor (alpha 2AAR) is polarized to the basolateral membrane of Madin-Darby canine kidney cells via direct targeting. Examination of mutant alpha 2AAR reveals that direct delivery is independent of NH2-terminal glycosylation, COOH-terminal acylation, or protein sequences within the large third cytoplasmic loop or COOH-terminal tail. Combined mutation of these structural features also does not perturb alpha 2AAR delivery, suggesting that a three-dimensional structure imparted by non-contiguous endofacial sequences does not confer alpha 2AAR targeting and that motifs in or near the bilayer must be involved in targeting of the alpha 2AAR. Mutation of a conserved Asp residue in transmembrane two that alters receptor-G-protein interactions also does not impair alpha 2AAR targeting. Finally, modification of sequences in transmembrane seven that resemble tyrosine-containing endocytosis motifs utilized for targeting by some proteins does not perturb alpha 2AAR sorting. Interestingly, deletion of the large third cytoplasmic loop of the alpha 2AAR decreases receptor half-life on the basolateral surface from approximately 11 to 4.5 h without altering the ability of the alpha 2AAR to couple to G-proteins. These data suggest that although targeting of the alpha 2AAR likely involves bilayer sequences, the third cytoplasmic loop may contain structural features that promote stabilization of the alpha 2AAR on the basolateral surface of Madin-Darby canine kidney cells.

The alpha 2A-adrenergic receptor is targeted directly to the basolateral membrane domain of Madin-Darby canine kidney cells independent of coupling to pertussis toxin-sensitive GTP-binding proteins.

The alpha 2-adrenergic receptor (alpha 2-AR) is a member of the seven transmembrane-spanning G-protein-coupled receptor superfamily. In the kidney, the alpha 2-AR is most abundant in the epithelial cells of the proximal tubule where it is important in enhancing Na+ reabsorption via the modulation of Na+/H+ exchange. Radioligand binding and physiological studies suggest that the alpha 2-AR residues primarily on the basolateral surface of these proximal tubule cells in vivo. To investigate the mechanisms underlying alpha 2-AR polarization in epithelial cells, we permanently expressed wild-type and an epitope-tagged version of the alpha 2A-AR in Madin-Darby canine kidney (MDCK) cells. Using a steady-state surface biotinylation assay, we observe that 80-90% of the alpha 2A-AR in MDCK cell clones is located on the basolateral membrane domain. Immunolocalization studies confirm the biotinylation results and demonstrate that the alpha 2A-AR is actually confined primarily to the lateral domain of the basolateral surface. Metabolic labeling experiments suggest that basolateral polarization of the alpha 2A-AR is achieved by direct targeting of the receptor to the basolateral domain. Targeting of the alpha 2A-AR to the basolateral surface is not perturbed by pertussis toxin-treatment of MDCK cells, suggesting that coupling of the alpha 2A-AR to GTP-binding proteins is not important for receptor polarization.

Mutations in the SH3 domain of the src oncogene which decrease association of phosphatidylinositol 3'-kinase activity with pp60v-src and alter cellular morphology.

To analyze the signaling pathways utilized in malignant transformation by pp60v-src, we have isolated and characterized src mutants which possess normal levels of protein tyrosine kinase activity but which cause only a partially transformed phenotype. Our hypothesis is that such mutants are partially defective for transformation because they are defective in their ability to activate specific components of the cellular signaling machinery while still activating others. In this communication, we report on the molecular and biochemical characterization of one such mutant, CU12 (D. D. Anderson, R. P. Beckmann, E. H. Harms, K. Nakamura, and M. J. Weber, J. Virol. 37:455-458, 1981). Cells infected with this mutant are capable of anchorage-independent growth, but rather than exhibiting the rounded and refractile morphology characteristic of wild-type-infected cells, they display an extremely elongated, fusiform morphology. The morphological properties of this mutant src could be accounted for entirely by a single mutation in the SH3 domain (lysine 106 to glutamate). Other mutations were constructed in this region by in vitro mutagenesis, both in a v-src and in an activated c-src background, and several of them also induced a fusiform morphology. All of the mutations inducing fusiform morphology also resulted in decreased association of pp60src with phosphatidylinositol 3'-kinase activity. In addition, association of pp60src with some tyrosine-phosphorylated proteins was altered. We propose that the SH3 domain participates (along with the SH2 domain) in the interaction of pp60src with cellular signaling proteins, and we speculate that the association with phosphatidylinositol 3'-kinase plays an important role in the regulation of cellular morphology.

Identification and characterization of the substance P receptor in sheep intestinal smooth muscle membranes.

Substance P (SP) is one of the endogenous tachykinin peptides implicated in neurogenic inflammation and may be critically involved in diseases as diverse as asthma, arthritis and inflammatory bowel disease. The current study was initiated to identify a rich source of SP receptor that would be amenable for studying the regulatory mechanism of the receptor. By using a radioligand receptor binding technique, sheep ileal smooth muscle membranes showed a much higher density of [3H]SP specific binding than other non-neural rat or sheep tissues and organs surveyed. Of the protease inhibitors tested, only phosphoramidon, a specific and potent enkephalinase inhibitor, prevented the degradation of [3H]SP and enhanced [3H]SP binding to the membrane. [3H]SP binding to the specific binding sites in the membranes was time-dependent and reached a steady state after 60 min at 22 degrees C in 25 mM Tris.NH3 (pH 7.4). Calcium and magnesium ions enhanced [3H]SP specific binding. Saturation binding studies showed that the dissociation constant (KD) and the density of maximum binding sites for [3H]SP specific binding were 0.54 nM and 83 fmol/mg of protein, respectively. The specificity of the [3H]SP labeled sites was SP greater than (4-11) SP greater than eledoisin greater than spantide greater than neurokinin-A greater than D-Pro2D-Phe7D-Trp9-SP. Neurokinin-B and senktide showed no inhibition of [3H]SP binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the soluble leukotriene B4 receptor from sheep lung membranes.

Leukotriene B4 (LTB4) is an arachidonate metabolite which elicits a variety of pro-inflammatory responses by activation of a guanine-nucleotide-binding protein-coupled membrane receptor. As a prelude to receptor isolation and purification, we have established assay methods for LTB4 receptor solubilization and characterization from sheep lung membranes. [3H]LTB4 binding to the soluble receptor was saturable, specific, protein-concentration- and time-dependent and reversible. Binding of [3H]LTB4 was enhanced by divalent cations and inhibited by sodium ions in a manner analogous to its binding to the human leukocyte membrane receptor. Saturation binding yielded a dissociation constant (Kd) of 0.50 +/- 0.05 nM and a receptor density (Bmax) of 330 +/- 90 fmol/mg of protein for [3H]LTB4 binding to detergent-solubilized receptor. In competition experiments, the rank order of binding affinity was LTB4 greater than 20-OH-LTB4 greater than trans-homo-LTB4 greater than 6-trans-LTB4 greater than U-75302. Gel-filtration chromatography showed that the LTB4 receptor protein in the detergent micellar state has a molecular mass in the range 800-1000 kDa. These results demonstrate that the physiologically and pharmacologically important LTB4 receptor may be readily solubilized from sheep lung membranes without alteration in binding specificity and characteristics, suggesting that sheep lung membranes represent a rich source with which to pursue receptor isolation and purification.

Protein kinase C: a new linkage marker for growth hormone and for COL1A1.

An expanded linkage group on the long arm of human chromosome 17 is reported. Using the CEPH panel of DNAs and restriction fragment length polymorphism (RFLP) markers for the centromere locus (D17Z1), growth hormone (GH1), collagen type I alpha 1 (COL1A1), and protein kinase C-alpha polypeptide (PKCA) loci, theta values of 0.03, 0.11, and 0.23 were found between PKCA and GH1, PKCA and COL1A1, and PKCA and D17Z1, respectively. The theta values calculated for GH1 versus COL1A1 or D17Z1 were 0.11 and 0.23, respectively. Sex-specific recombination rates were calculated for the best likelihood order and demonstrate female recombination greater than male recombination. Therefore, the loci studied span a map region of approximately 30 cm between 17cen and 17q24, with the most likely gene order being D17Z1-COL1A1-PKCA-GH1.

Bradykinin analogs antagonize bradykinin-induced second messenger production in a sensory neuron cell line.

Bradykinin is the prime initiator of pain and the key initial activator of the inflammatory response at the site of tissue injury. The subsequent transfer of nociceptive information (pain sensation) into the central nervous system is then mediated via afferent type C dorsal root ganglion neurons. A recently developed hybrid cell line, F-11, shows many qualities characteristic of these pain-sensitive cells. In these neuronal hybrids, we have found that bradykinin induces sequential elevation in the concentrations of several second messengers involved in neuronal activation, including inositol trisphosphate (6.5-fold), intracellular calcium (2.7-fold), and cyclic GMP (20.5-fold). Importantly, the production of these second messengers is potently inhibited by several novel bradykinin antagonists that possess no intrinsic agonist activity. The same relative rank order of potency of inhibition of bradykinin-induced second messenger production was achieved in the inositol trisphosphate, calcium, and cyclic GMP assay systems, suggesting strongly that all three messenger systems are being activated by the same bradykinin receptor. The most potent antagonist was D-Arg0-Hyp3-Thi5,8-D-Phe7-bradykinin, which inhibited in a competitive manner, with pA2 values, upon Schild plot analysis, in the nanomolar range. These potent bradykinin antagonists may be useful in the characterization of bradykinin receptors and in the clinical management of pain and inflammation.

Asparagine catabolism in rat liver mitochondria.

A large portion of mitochondrial asparagine (Asn) is degraded by asparagine amino-transferase to produce alpha-ketosuccinamate (alpha KSA), which is then hydrolized by omega-amidase to produce oxaloacetate (OAA) and ammonia. This is in contrast to the catabolism in the cytosol, where the main catabolic route for Asn occurs initially via asparaginase-catalyzed hydrolysis to form aspartate and ammonia. Mitochondrial production of OAA from Asn was followed by monitoring the decrease in the rate of succinate oxidation (which is inhibited by OAA) in both coupled and uncoupled mitochondria. Rapid OAA production was found to be dependent on the presence of both Asn and glyoxylate, and was eliminated by the aminotransferase inhibitor, aminooxyacetate (AOX). HPLC separation and quantitation of alpha-keto acids and amino acids allowed direct observation of the proposed mitochondrial pathway. Studies using L-[U-14C]Asn in mitochondria yielded labeled carbon in alpha KSA, OAA, and CO2 when either an alpha-keto acid or glyoxylate was provided. The extent of the labeled carbon in these products was greatly influenced by factors that affected the citric acid cycle and oxidative phosphorylation. Carbon dioxide production from Asn alone, even in the presence of AOX, suggested the existence of at least one additional Asn catabolic pathway in the rat liver mitochondria which does not involve alpha KSA as an intermediate.

Should the patella be resurfaced in total knee arthroplasty? Efficacy of patellar resurfacing.

To assess the long-term efficacy of patellar resurfacing, 100 knees were evaluated in 84 patients. The operations were performed between 1978 and 1982. The follow-up period ranged from 60 to 103 months. The diagnosis was degenerative joint disease (DJD) in 83%, rheumatoid arthritis in 12%, and miscellaneous in 5% of the knees. The implant (47 knees) and nonimplant (53 knees) groups were comparable with respect to age, body size, and length of follow-up period. The analysis revealed equivocal results. Considering all diagnostic categories combined, rest pain was marginally better in the resurfaced group (p = 0.04), but this difference resulted from an unequal distribution of subjects between mild and zero pain categories. Pain with walking, maximum walking distance, ability to climb stairs and rise from a chair, active arc of motion, extensor lag, and quadriceps strength were similar in the two groups. When the DJD group was considered separately, no significant difference emerged. There was little evidence to support a recommendation for routine patellar resurfacing in total knee arthroplasty.

Ca2+/calmodulin-dependent protein kinase II. Identification of a regulatory autophosphorylation site adjacent to the inhibitory and calmodulin-binding domains.

Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) autophosphorylated under limiting conditions (7 microM [gamma-32P]ATP, 500 microM magnesium acetate, 4 degrees C) was analyzed by CNBr cleavage and peptide mapping to determine the site of autophosphorylation that brings about transition of the kinase to the Ca2+-independent form. Reverse phase high performance liquid chromatography (HPLC) (C3) revealed one major CN-Br 32P-peptide (CB1) that eluted at about 6% propanol. This peptide contained [32P]threonine, but almost no [32P]serine, and migrated as a single band (Mr = 3000-3500) in polyacrylamide gels run in the presence of urea and sodium dodecyl sulfate. The properties of CB1 were compared to the properties of a 26-residue synthetic peptide containing the CaM-binding and inhibitory domains as well as a consensus phosphorylation sequence (-Arg-Gln-Glu-Thr-) of rat brain CaM-kinase II (residues 282-307 and 283-308 of the alpha and beta subunits, respectively). CB1 and the synthetic peptide comigrated in urea/sodium dodecyl sulfate gels, co-eluted from reverse phase HPLC (C3 and C18) and from Sephadex G-50, and exhibited Ca2+-dependent calmodulin-binding properties. When the two peptides were subjected to automated Edman sequence analysis, both exhibited a burst of 32P release at cycle 5, which is consistent with the expected amino-terminal sequence of the two peptides, i.e. His-Arg-Gln-Glu-Thr(PO4)-. These findings indicate that autophosphorylation of Thr286 (alpha subunit) and Thr287 (beta subunit) is responsible for transition of CaM-kinase II to the Ca2+-independent form.

Separation of citric acid cycle intermediates by high-performance liquid chromatography with ion pairing.

A method to separate underivatized tricarboxylic acid cycle intermediates within 20 min using the commonly available C15 high-performance liquid chromatography column has been developed. Ion pairing using tetrabutylammonium cations and isocratic conditions is used to separate the intermediates which are then detected at 210 nm. Separation was optimized by altering pH, the concentration of sodium sulfate and the pairing ion. This technique permits the detection of as little as 120 nmol of citrate to 0.5 nmol of fumarate. Physiological samples of rat liver mitochondria, human urine, and orange juice were analyzed.

Amino acid content of L5178Y and L5178Y/L-ASE cells after L-asparaginase treatment.

The amino acid contents of tumor cells that are either sensitive or resistant to treatment with L-asparaginase were measured. These amino acid concentrations were measured as a function of incubation time with L-asparaginase or as a function of the L-asparaginase dose. The cell types compared were the mouse leukemia lines L5178Y (sensitive to L-asparaginase treatment) and L5178Y/L-ASE (resistant to L-asparaginase treatment). Upon L-asparaginase treatment both cell lines lost most of their cellular asparagine but, whereas the resistant cells exhibited the ability to rebound to about 50% of initial values, the sensitive cells did not. While previous work had suggested that asparagine-dependent glycine synthesis was essential for sensitive cells (but not in resistant cells), we found no difference in the glycine content of either of the two cell lines as a function of either time or dose that would support this hypothesis. Major differences between the two cell lines were seen in the content of the essential amino acids before treatment with L-asparaginase. After incubation without L-asparaginase the contents of the two cell lines became similar. These results are discussed in terms of possible mechanisms of L-asparaginase sensitivity and resistance.

Comparison of glycine metabolism in mouse lymphoma cells either sensitive or resistant to L-asparaginase.

Previous work suggested a relationship between glycine metabolism and the effect of L-asparaginase upon tumor cells. Therefore, L5178Y (sensitive) or L5178Y/L-ASE (resistant) ascites lymphoma cells were incubated with 14C-labeled glyoxylate, glycine, serine, or asparagine, and the metabolism to other amino acids was measured by high performance liquid chromatography. Metabolic differences between the two cells lines were found. Under control conditions, the interconversion rate of glycine and serine via serine hydroxymethyltransferase (SHMT) was higher in sensitive than in resistant cells. The transformation rate of glyoxylate to serine was also higher in sensitive cells. These results may indicate a difference in the activity of SHMT. An alternate explanation would be that transport or diffusion of serine and glycine into sensitive cells is greater than into resistant cells. Several crucial metabolic differences were observed between the two cell types when L-asparaginase was added. A key difference is the decrease of glycine synthesis from glyoxylate observed in the sensitive cells compared to resistant cells which show no change. This suggests that asparagine is used for transamination of glyoxylate. Also, only sensitive cells appear to compensate for L-asparaginase-induced loss of glycine formation from glyoxylate by increasing glycine synthesis from serine. Alterations in sensitive tumor glycine metabolism may be an important function of L-asparaginase anticancer activity.

Noncardiogenic pulmonary edema and invasive cardiovascular monitoring.

When a parturient develops clinical and roentgenographic evidence of pulmonary edema during labor or delivery, the obstetric staff often concludes that iatrogenic overhydration or left ventricular failure is the cause. This impression may be reinforced by increased central venous pressure measurements. Although acute respiratory failure (ARF) (non-cardiogenic pulmonary edema) is well described in the medical and surgical literature, the diagnosis is rarely, if ever, made in the obstetric patient unless aspiration pneumonitis is suspected. Unfortunately the usual historic and clinical findings do not differentiate ARF from cardiogenic pulmonary edema. The diagnosis of ARF is based on the finding of pulmonary edema in the absence of an elevated pulmonary capillary wedge pressure. As invasive cardiovascular monitoring is not usually part of the obstetrician's armamentarium, many cases of ARF may be mislabeled and mistreated as cardiogenic pulmonary edema. Four illustrative cases are presented.