Externally suppressible proline quadruplet ccc U.

Three (+1) frameshift mutations located at different genetic sites respond with high specificity to the same external suppressor. In each case, the suppressor restores small amounts of protein that is normal in electrophoretic mobility and heat stability. One of these proteins has been shown to have the wildtype amino acid sequence. The messenger RNA quadruplet CCCUappears to be common to all three frameshift sites and to be translated by the suppressor as proline. A likely suppressor agent is a proline transfer RNA with a quadruplet anticodon or its functional equivalent.

Rapid in situ cytochemical classification of CFU-C colonies in soft agar culture: permanent whole culture preparations.

Soft agar culture CFU-C (colony-forming units in culture) are rapidly classified in situ as eosinophil, macrophage, and neutrophil-monocyte types by whole culture staining with luxol fast blue for eosinophil specific granules and acetoorcein for nuclei. Stained colonies may be picked and examined individually as wet mount cover slip preparations, or the agar culture may be air dried and mounted permanently. The whole culture stain has been variously modified for the enzyme markers alpha-naphthyl acetate esterase and peroxidase and for nuclear staining alone with acetoorcein.

Monocyte nonspecific esterase. Enzymologic characterization of a neutral serine esterase associated with myeloid cells.

Monocytes contain a characteristic, prominent set of membrane-bound nonspecific esterases with a slightly acid isoelectric point. These esterases are also detected at modest levels in some granulocyte preparations. They are not apparent in lymphocytes. Among 18 fresh myeloid leukemias and myeloid leukemia cell lines, those of subtypes M4 (myelomonocytic) and M5 (monocytic) were strongly positive; some of subtypes M1-M3 (granulocytic) were moderately positive. The esterases were not detected among 32 fresh lymphoid leukemias and lymphoid leukemia and lymphoblast cell lines. The membrane-bound monocyte esterases, solubilized by treatment of monocyte preparations with nonionic detergent, were resolved by ion-exchange chromatography. The monocyte species account for 80-95% of the total nonspecific esterase activity of monocytes. The resolved enzymes behave as neutral serine carboxyl esterases and are highly sensitive to inhibition by diisopropylfluorophosphate (DFP) and also by sodium fluoride. Similar analysis of a lymphocyte preparation yielded no detectable monocyte esterases, but yielded numerous other forms which were generally resistant to inhibition by DFP and NaF. These nonspecific esterases are also present at background levels in monocytes. The resolution and characterization of the membrane-bound serine esterases from monocytes demonstrates the basis for the well-known cytochemistry of monocytes. The results are also crucial to the development of an immunologic surface marker test for myeloid cells and the study of monocyte membrane physiology.

A recombinant clone of HIV-1 preferentially transmitted in normal peripheral blood mononuclear cells.

To study biologic properties associated with specific regions of HIV-1, a chimera, pHX-JY1, was constructed by exchanging the vif-env region of a Zairian molecular clone (JY1) with that of pHXB2gpt, a full-length biologically active proviral clone of North American origin. Virus was produced by transfection of permissive cells with parental and recombinant clones, and the biologic and molecular properties of these viruses were compared. Virus derived from pHXB2gpt infected phytohemagglutinin (PHA)-activated normal peripheral blood mononuclear cells (PBMC) and CD4+ leukemic T cell lines equally well. In contrast, virus derived from pHX-JY1 was transmitted slowly to both PBMC and cell lines, and the infectivity of pHX-JY1 virus was two orders of magnitude greater for PBMC than for T cell lines. All essential viral genes in the exchanged JY1 vif-env region were intact and functioned comparably to those of the parent clone in transfected COS-1 cells. The findings suggest differences in these regions of the HIV-1 genome may play an important role in differential cell tropism.

Nucleotide sequence analysis of the env gene of a new Zairian isolate of HIV-1.

As a further step in the continuing process of defining the extent and nature of variability of the envelope (env) gene of HIV-1, we have cloned a new Zairian isolate, JY1, and sequenced the env gene of this isolate. Although the restriction map of the env region of JY1 was found to be more similar to that of the American prototype, BH10, than maps of all previously reported Zairian isolates and some American isolates, nucleotide sequencing of the JY1 env gene showed that it is among the most divergent from BH10 yet reported and that it differs from previously reported Zairian isolates almost to the same extent that it differs from BH10. A typical pattern of variable and constant regions was seen. A number of complex duplications were found in the hypervariable regions of JY1. The unique and highly divergent nature of the env gene of JY1 enhances its usefulness as part of a panel of HIV-1 isolates being evaluated in biologic and immunologic studies toward vaccine development.

Rapid amylase and lipase determinations by nephelometry.

The Coleman 91 nephelometer provides rapid, simple amylase and lipase assays, which are particularly suited to emergency requests. Linearity of amylase compares favorably with that of the Phadebas assay, and comparable precision was obtainable with serum and urine. Normal ranges for serum amylase are slightly higher than those based on the amyloclastic end-point assay. The serum lipase assay shows improved linearity over titrimetric procedures, although kinetics remain variably non-linear. Occasional sera show discordantly elevated nephelometric lipase and normal titrimetric lipase. Precision of the nephelometric lipase assay is somewhat lower than that of amylase; normal ranges are considerably higher than those based on titrimetry. Extremely lactescent sera may yield falsely low nephelometric amylase and lipase activities: these sera must be serially diluted to achieve actual values.

Transiently elevated apparent lipase by nephelometry.

Occasional patient sera showing normal or minimally elevated lipase activity in the 6-hour titrimetric assay and discordantly high lipase activity in the 6-minute nephelometric assay were encountered. Most of these sera had normal amylase activity. They represented about 1% of outpatient sera subjected to amylase assay. A few sera with discordant lipase activities showed elevated amylase activities and were from patients with the diagnosis of pancreatic dysfunction. Nephelometric lipase assay in these sera showed pronounced nonlinear kinetics; in the most discordant cases a gradual decay of initially high activity to zero occurred. Elevated nephelometric lipase activity was lost inordinately in serial dilution of serum or on serial reduction of undiluted assay volume. In tracer experiments, two of these sera liberated no significant amount of free fatty acids after extensive "de-emulsification" of the nephelometric substrate mixture. This nonlipolytic "deemulsifying" activity was found to be relatively heat-stable in one serum. The nature of this activity remains obscure. A simple and effective protocol is suggested to detect and identify these aberrant nephelometric lipases in the routine laboratory.

Monocyte nonspecific esterase: purification and subunit structure.

Monocyte nonspecific esterase has been purified from cultured cells of the acute myeloid leukemia cell line, ML-1. The purified enzyme shows the characteristic properties of the monocyte neutral serine carboxyl esterase, with high sensitivity to organophosphorus inhibitors and sodium fluoride inhibitor. The enzyme is a membrane protein which in the native state exists as a monomer of a mol wt of approximately 68,000 and a trimer of mol wt 205,000. These forms exhibit a complex pattern of dissociation and reassociation based on apparent noncovalent binding of subunits. The delipidated dissociated enzyme runs as a single protein chain of a mol wt of approximately 62,000 on sodium dodecyl sulfate (SDS) gel electrophoresis. The relation of the subunits to monocyte isoenzymes seen on isoelectric focusing (IEF) and polyacrylamide gel electrophoresis at pH 9.5 (pH 9.5 PAGE) of cell extracts is demonstrated. Availability of purified enzyme allows development of monoclonal antibodies and analysis of myeloid differentiation. In addition, the substrate specificity and function of the purified monocyte ectoenzyme are being examined.

Direct polymerase chain reaction for detection of human immunodeficiency virus in blood spot residues on filter paper after elution of antibodies: an adjunct to serological surveys for estimating vertical transmission rates among human immunodeficiency virus antibody-positive newborns.

Blood spot residues on filter paper circles from which antibodies have been eluted remain suitable for follow-up polymerase chain reaction to detect human immunodeficiency virus (HIV) provirus. A method has been developed for specimen processing and nested polymerase chain reaction amplification with a single tube. HIV-positive specimens can be detected by simple, direct electrophoresis of amplified material to a single copy of provirus. The procedure is particularly suited for survey purposes to estimate rates of vertical transmission of virus among HIV antibody-positive newborns, in whom virus loads may be low.

A method for nested PCR with single closed reaction tubes.

Toward the goal of reducing diagnostic false positives while retaining high sensitivity, a closed-tube nested PCR procedure has been developed for detecting low-copy-number human immunodeficiency virus (HIV) gag target DNA sequences. Master mix for amplification 2, in a hanging gel matrix at the reaction tube top, remains sequestered from the reaction space of the tube during amplification 1. A severalfold excess of inner over outer primers is built into the procedure to assure the high sensitivity of nested PCR. The master mix for amplification 2 is then introduced into the reaction space by centrifugation, and the second amplification is performed as usual. The closed-tube nested procedure shows sensitivity approaching that of the open-tube control procedure, which detects a single copy of HIV gag target DNA at near-theoretical frequency, typically with microgram yields of specific amplification product.

Nonspecific esterases of the formed elements: zymograms produced by pH 9.5 polyarcrylamide gel electrophoresis.

The formed elements of human blood each contain multiple isoenzymes of nonspecific esterase that hydrolyze short chain alpha naphthyl esters. Zymograms that are characteristic of each type of formed element are obtained by subjecting purified preparations of each to polyacrylamide slab gel electrophoresis at pH 9.5 and subsequent staining of the gels for esterase activity. The most prominent isoenzyme detected is a species of low mobility that is reactive with either acetyl or butyryl esters and is highly sensitive to inhibition by 40 mM sodium fluoride. Also detected are several major acetyl esterases and a single butyryl esterase, all of which are relatively fluoride resistant. The intercellular distribution of isoenzymes varies from element-specific to pancellular. The prominent fluoride-sensitive acetyl, butyryl esterase, is the major isoenzyme of monocyte zymograms, which is consistent with the well known cytochemistry of monocytes. Lesser but significant amount (2%-3% of monocyte levels) of this isoenzyme were also detected in granulocyte zymograms. This system may prove useful in the study of differentiation of blood cells and in the classification of acute leukemias.

Nature of the hisD3018 frameshift mutation in Salmonella typhimurium.

Histidinol dehydrogenase from three differing revertants of ICR-191A-induced frameshift hisD3018 has been purified and examined for amino acid replacements. The enzyme from one spontaneously arising revertant, R7, contains an extra proline residue, whereas that from another, R5, contains an extensive frameshifted sequence, four amino acid residues of which have been identified to date. The amino acid replacement data are in agreement with the in vitro code word assignments and allow the characterization of the hisD3018 frameshift as an addition of one nucleotide pair, most likely guanine plus cytosine. Enzymatic data for those ICR-191A-induced revertants of hisD3018 arising within the hisD gene indicate that the enzyme is wild type and, therefore, that ICR-191A can cause deletions as well as additions of single base pairs. The wild-type amino acid sequence is restored in enzyme from an N-methyl-N'-nitro-N-nitrosoguanidine (NG)-induced revertant, R29, suggesting that NG is a base-deleting as well as a base-substituting mutagen. The unusual response of hisD3018 to external suppressors is considered in terms of reinitiation of protein synthesis out of phase, coupled with suppression of a nonpermissive missense codon so generated, and of an alternative hypothesis invoking a true frameshift suppressor transfer ribonucleic acid with an extended or deleted anticodon.