Syntheses of (Z)-and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid and their inactivation of gamma-aminobutyric acid aminotransferase.

(Z)- and (E)-4-amino-2-(trifluoromethyl)-2-butenoic acid (4 and 5, respectively) were synthesized and investigated as potential mechanism-based inactivators of gamma-aminobutyric acid aminotransferase (GABA-AT) in a continuing effort to map the active site of this enzyme. The core alpha-trifluoromethyl-alpha,beta-unsaturated ester moiety was prepared via a Reformatsky/reductive elimination coupling of the key intermediates tert-butyl 2,2-dichloro-3,3,3-trifluoropropionate and N,N-bis(tert-butoxy-carbonyl)glycinal. Both 4 and 5 inhibited GABA-AT in a time-dependent manner, but displayed non-pseudo-first-order inactivation kinetics; initially, the inactivation rate increased with time. Further investigation demonstrated that the actual inactivator is generated enzymatically from 4 or 5. This inactivating species is released from the active site prior to inactivation, and as a result, 4 and 5 cannot be defined as mechanism-based inactivators. Furthermore, 4 and 5 are alternate substrates for GABA-AT, transaminated by the enzyme with Km values of 0.74 and 20.5 mM, respectively. Transamination occurs approximately 276 and 305 times per inactivation event for 4 and 5, respectively. The enzyme also catalyzes the elimination of the fluoride ion from 4 and 5. A mechanism to account for these observations is proposed.     

Treatment of methanol poisoning with ethanol and hemodialysis.

Twelve cases of methanol poisoning are reviewed. The clinical presentation and biochemical features are described and the results of treatment with alkali, ethanol and dialysis reported. The outcome of methanol poisoning appears to be related more to the interval between the time of ingestion and the start of therapy and to the degree of acidosis than to the initial serum methanol level. Therefore, early and aggressive treatment with bicarbonate and ethanol and subsequent institution of hemodialysis are strongly recommended whenever methanol can be detected in the blood, especially when metabolic acidosis of the anion-gap type is present, when mental or visual disturbances are present, or when more than 30 ml of absolute methanol has been consumed.     

Analytical errors in measuring radioactivity in cell proteins and their effect on estimates of protein turnover in L cells.

Previous studies from this laboratory on protein turnover in 3H-labelled L-cell cultures have shown recovery of total 3H at the end of a 3-day experiment to be always significantly in excess of the 3H recovered at the beginning of the experiment. In this study we have critically reviewed a number of possible sources for this error in measuring radioactivity in cell proteins. 3H-labelled proteins, when dissolved in 0.3 M-NaOH and counted for radioactivity in a liquid-scintillation spectrometer, showed losses of 30-40% of the radioactivity; neither external or internal standardization compensated for this loss. Hydrolysis of these proteins with either Pronase or concentrated HCl significantly increased the measured radioactivity. In addition, approx. 5-10% of the cell protein is left on the plastic culture dish when cells are recovered in phosphate-buffered saline. To aggravate this latter loss further, this surface-adherent protein, after pulse labelling, contains proteins of high radioactivity that turn over rapidly and make a major contribution to the accumulating radioactivity in the medium. These combined errors can account for up to 60% of the total radioactivity in the cell culture. Similar analytical errors have been found in studies of other cell cultures. The effect of these analytical errors on estimates of protein turnover in cell cultures is discussed.     

Fine needle aspiration cytology of neoplasms metastatic to the breast

The fine needle aspiration (FNA) cytologic findings in 18 cases of metastatic neoplasms of the breast are reported. The cases were encountered in a combined series of 2,529 FNA breast biopsies, of which 666 were malignant; the metastatic neoplasms of the breast thus constituted 2.7% of all the malignant breast tumors. The series consists of 15 women and 3 men, with a mean age of 48 years (range of 11 to 73 years). Sixteen biopsies confirmed metastatic malignancy in patients with known extramammary primaries; the prebiopsy clinical diagnoses in six of the patients were benign breast lesions. In eight patients, the clinical differential diagnosis was either a benign or malignant primary breast lesion versus a metastatic malignancy. In two additional patients, the FNA biopsy identified metastatic neoplasms from unsuspected extramammary primaries. The metastatic neoplasms included three small-cell carcinomas of the lung, one squamous-cell carcinoma of the lung, two malignant melanomas, three ovarian malignancies, including a dysgerminoma, and one each of carcinoma of the fallopian tube, endometrial carcinoma, transitional-cell carcinoma of the urinary bladder, prostatic carcinoma, acute granulocytic leukemia, lymphoma, mycosis fungoides, hepatoma and neuroblastoma of the retroperitoneum. Recognition of unusual cytologic patterns raised the suspicion of, or confirmed the diagnosis of, malignancy in all cases, with no false-negative diagnoses. None of the cases were cytologically interpreted as a primary breast malignancy. Ancillary studies performed on the FNA material, including immunocytochemistry, contributed to a definitive diagnosis in three cases. FNA diagnosis of metastatic malignancy of the breast is essential in order to avoid unnecessary mastectomy and to ensure appropriate chemotherapy and/or irradiation treatment.     

Analysis of the harmonic content of the tidal flow waveforms in infants.

The aim of this study was to examine whether the spectral characteristics of tidal flow waveform reflect the interaction between the control of breathing and lung mechanics in 10 healthy infants (H), 10 infants with a history of wheezing disorders (W), and 10 infants with chronic lung disease (CLD). From the flow waveform, we calculated a shape index, the harmonic distortion (k(d)), which quantifies the extent to which a periodic signal deviates from a sine wave. The k(d) of the entire tidal flow waveform did not significantly discriminate between diagnostic groups. However, k(d) was sensitive to maturation: it increased from 0.26 at 1 mo to 0.37 at 6 mo of age (P < 0.002). Furthermore, the frequency (f) spectra of the flow (V) amplitudes between 0.13 and 10 Hz followed a power law: V(f) approximately f(-s), where s (slope) is the exponent in the power law. The exponent of the healthy infants s(H) was 4.24 [95% confidence interval (CI) = 0.2] at 1 mo, 4.39 (CI = 0.16) at 6 mo, and 4.35 (CI = 0.19) at 12 mo and not significantly changing with age. The mean value of s(W) was marginally lower (4.09 +/- 0.28; P < 0.05) than that of s(H). The mean s(CLD) was significantly lower (3.04 +/- 0.31; P < 0.001). Lower values of s and higher values of k(d) indicate an increased complexity of the feedback mechanisms determining tidal flow waveform and may be associated with disease.     

Carbonic Anhydrase and the Uptake of Inorganic Carbon by Synechococcus sp. (UTEX-2380)

We report the changes in the concentrations and ¹⁸O contents of extracellular CO₂ and HCO₃⁻ in suspensions of Synechococcus sp. (UTEX 2380) using membrane inlet mass spectrometry. This marine cyanobacterium is known to have an active uptake mechanism for inorganic carbon. Measuring ¹⁸O exchange between CO₂ and water, we have found the intracellular carbonic anhydrase activity to be equivalent to 20 times the uncatalyzed CO₂ hydration rate in different samples of cells that were grown on bubbled air (low-CO₂ conditions). This activity was only weakly inhibited by ethoxzolamide with an I₅₀ near 7 to 10 micromolar in lysed cell suspensions. We have shown that even with CO₂-starved cells there is considerable generation of CO₂ from intracellular stores, a factor that can cause errors in measurement of net CO₂ uptake unless accounted for. It was demonstrated that use of ¹³C-labeled inorganic carbon outside the cell can correct for such errors in mass spectrometric measurement. Oxygen-18 depletion experiments show that in the light, CO₂ readily passes across the cell membrane to the sites of intracellular carbonic anhydrase. Although HCO₃⁻ was readily taken up by the cells, these experiments shown that there is no significant efflux of HCO₃⁻ from Synechococcus.     

Involvement of the 2'-5' A pathway in the augmentation of natural killer activity

Pretreatment of human large granular lymphocytes (LGL) or unseparated peripheral blood mononuclear cells with interferon (IFN) resulted in a significant augmentation of natural killer (NK) activity. This increase was paralleled by an increase in the 2'-5'A synthetase activity. In order to investigate the possibility that IFN might be inducing augmentation of NK cells via the 2'-5'A pathway, we tested the effects of nonphosphorylated core material [(A2'p)2A] and of the triphosphorylated form of the 2'-5'A [ppp(A2'p)2A]. The core material had no detectable effect on NK activity. In contrast, when experiments were performed with the triphosphorylated form of 2'-5'A, NK activity was stimulated. In order to achieve activation, permeabilization of LGL with calcium chloride was necessary and, under these conditions, a dose-dependent augmentation of NK activity was seen. However, the calcium treatment had considerable toxic effects on basal levels of NK activity. Collectively, these results suggest that IFN may be inducing augmentation of NK activity via the 2'-5'A pathway. Further studies will be necessary to determine the effects of IFN and/or 2'-5'A on subsequent activation steps in the process leading to cytotoxicity by NK cells.     

Genetic analysis of human hypoxanthine-guanine phosphoribosyltransferase deficiency

Hypoxanthine-guanine phosphoribosyltransferase (HPRT; IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) functions in the purine-metabolic salvage pathway. Two clinical syndromes are associated with a deficiency in HPRT enzyme activity. Virtually complete deficiency leads to the Lesch-Nyhan syndrome, whereas partial deficiency results in hyperuricemia and severe gouty arthritis. Marked heterogeneity in the mutations leading to HPRT deficiency has been found. Mutant enzymes vary with respect to levels of HPRT immunoreactive protein, electrophoretic migration, kinetic properties and amino acid sequence. Analysis of DNA and RNA from patients with HPRT deficiency has revealed point mutations, an internal gene duplication and partial as well as complete gene deletions accounting for the various HPRT mutant enzymes.     

Direct Evidence for Changes in the Resistance of Legume Root Nodules to O2 Diffusion

Indirect evidence suggests that legumes can adjust rapidly theresistance of their root nodules to O₂ diffusion. Here we describeexperiments using O₂ specific micro-electrodes and dark fieldmicroscopy to study directly the operation of this diffusionbarrier. The O₂ concentration sensed by the electrode decreasedsharply in the region of the inner cortex and was less than1.0 mmol m^–3 throughout the infected tissue in nodulesof both pea (Pisum sativum) and french bean (Phaseolus vulgaris).In a number of experiments the ambient O₂ concentration wasincreased to 40% while the electrode tip was just inside theinner cortex. In 13 out of 21 cases the O₂ concentration atthis position either remained low and unchanged or increasedirreversibly to near ambient values. In the remaining casesthe O₂ concentration increased after 1 to 2.5 min and then decreasedto its former value. These results are ascribed to an increasein resistance of the barrier in response to increased O₂ fluxinto the nodule. It was shown microscopically that air spacesboth at the boundary between the infected zone and the innercortex, and within the infected zone started to disappear 3min after nodules were exposed to high ambient O₂ concentrationsand had disappeared completely after 8 min. These spaces werenot changed by exposure of the nodule for 10 min to either N₂or air. Key words: Oxygen, root nodules, air spaces     

Population genetics of coagulant factor IX: frequencies of two DNA polymorphisms in five ethnic groups.

Two frequently used restriction-enzyme polymorphisms (RFLPs) of coagulant F.IX, TaqI and XmnI, have been examined in five ethnic groups: white Americans, black Americans, East Indians, Chinese, and Malays. There is a distinct "cline" in the frequencies of both polymorphisms, from white Americans to Malays. The rarer type 2 alleles of both polymorphisms, in which middle recognition sites are present--and which in our sample reach their highest frequencies in white Americans--are marginally higher in four groups of Europeans previously reported by others. The frequencies of the rarer alleles are significantly higher in Europeans than in black Americans and East Indians, and these alleles are essentially absent in Chinese and Malays. The frequency of heterozygosity diminishes in the same order, being zero in Malays for both polymorphisms. The polymorphisms are in strong linkage disequilibrium, and in all groups the type 1 allele for TaqI is disproportionately accompanied by the type 1 allele for XmnI. The paucity of type 2 alleles and the low rate of heterozygosity in four non-European groups suggest that the polymorphisms will be of little diagnostic value south of Gibraltar and east of Suez. This prediction is confirmed by the observed haplotype frequencies in the black American and the Oriental groups.