Intracellular fusion of sequentially formed endocytic compartments

A polyclonal anti-fluorescein antibody (AFA) which quenches fluorescein fluorescence has been used to distinguish between two models of intracellular vesicle traffic. These models address the question of whether sequentially endocytosed probes will mix intracellularly or whether they are carried through the cell in a sequential, isolated manner. Using transferrin (Tf) as a recycling receptor marker, we incubated Chinese hamster ovary (CHO) cells with fluorescein-Tf (F-Tf) which is rapidly endocytosed. After the F-Tf was completely cleared from the surface, AFA was added to the incubation medium and entered endocytic compartments by fluid phase endocytosis. Fusion of a vesicle containing AFA with the compartment containing F-Tf results in binding of AFA to fluorescein and the quenching of fluorescein fluorescence. When AFA was added to the culture medium 2 min after clearance of F-Tf from the surface, time dependent fluorescence quenching occurred. After 20 min, 67% saturation of F-Tf with AFA was observed. When the interval between F-Tf clearance and AFA addition was increased to 5 min only 41% saturation of F-Tf was found. These data indicate that there are some compartments which are accessible for mixing with subsequently endocytosed molecules, but the efficiency of mixing falls off rapidly as the interval between pulses is increased. In CHO cells Tf swiftly segregates to a collection of vesicles or tubules in the para-Golgi region, and at steady state most of the F-Tf is in this compartment. Using digital image analysis to quantify quenching in this region, we have found that F-Tf/AFA mixing is occurring either within this compartment or before transferrin enters it.     

Primary cutaneous adenoid cystic carcinoma: a case report and review of the literature.

A case of adenoid cystic carcinoma of the ear in a 67-year-old man is presented along with a review of the literature. Adenoid cystic carcinoma most commonly arises in the salivary glands. Primary cutaneous adenoid cystic carcinoma occurs principally in the scalp and chest. Perineural invasion is seen in at least half the cases and is associated with an increased recurrence rate. Treatment consists of wide local resection with tumor-free margins. Just over half the patients will develop recurrence, with long tumor-free intervals necessitating long-term follow-up. Distant metastases are rarely seen. Radiation therapy is not curative and should be reserved for palliation.     

Linear, Single-Stranded Deoxyribonucleic Acid Isolated from Kilham Rat Virus

Kilham rat virus (KRV) was grown in a rat nephroma cell line and was purified by two isopycnic centrifugations in cesium chloride. The virus contains single-stranded deoxyribonucleic acid (DNA) with a molecular weight of approximately 1.6 x 10(6). The DNA was extracted from the virion by both phenol extraction and by 2% sodium dodecyl sulfate at 50 C. KRV DNA, extracted by both procedures, was observed in an electron microscope by using a cytochrome c or diethylaminoethyldextran monolayer. The DNA was also exposed to exonuclease I, an enzyme which hydrolyzes specifically linear, single-stranded DNA. Hydrolysis of 70 to 80% of the DNA was observed. Both the enzymatic and the electron microscope studies support the conclusion that extracted KRV DNA is a single-stranded, linear molecule. The length of the DNA was measured in the electron microscope and determined to be 1.505 +/- 0.206 mum.     

Metabolic Properties of Early and Late Vaccinia Virus Messenger Ribonucleic Acid

In vaccinia-infected cells, 60% of the viral messenger ribonucleic acid (mRNA) was associated with polyribosomes, and the remainder sedimented in a broad peak in the 30 to 74S region. The quantity of mRNA in polyribosomes was sharply reduced late in the infectious cycle [9 hr postinfection (PI)] to less than 30% of the 2-hr value. However, protein synthesis proceeded at a nearly constant rate from 2 to 13 hr PI. This ability of small quantities of late mRNA to support as much protein synthesis as do the much larger quantities of early mRNA was not due to an increase in stability, since late mRNA decays with a half-life of 13 min, whereas early mRNA has a half-life of 120 min. A similar decrease in viral mRNA synthesis without an accompanying decrease in viral protein synthesis was observed when deoxyribonucleic acid synthesis is inhibited. In contrast to the rapid decay of the late mRNA which was present in polyribosomes, the mRNA which sedimented in the 30 to 74S region remained unchanged even after a 2-hr period of exposure to actinomycin. The rate at which infected cells lose the capacity to synthesize specific viral proteins after exposure to actinomycin D was consistent with the half-life values of early and late mRNA that were observed.     

Sequential Formation of Vaccinia Virus Proteins and Viral Deoxyribonucleic Acid Replication

In vaccinia virus-infected cell cultures, cellular protein synthesis was inhibited 50% at 2 hr postinfection (PI) and 80 to 90% by 4 hr PI. Input virus was responsible for this inhibition. Five early proteins, coded for by the viral genome, could be detected at 2 to 3 hr PI. Normally, their synthesis did not continue beyond 6 hr PI, at which time synthesis of a different set of proteins began. When DNA replication was blocked, synthesis of these early proteins continued until 9 to 12 hr PI. The bulk of the proteins which were incorporated into mature virus were synthesized at 8 hr PI and thereafter. The time of their formation was close to the time at which virus maturation occurred. However, 15% of the protein found in mature virus was synthesized early in the infectious cycle. The quantity of “early viral protein” which was not incorporated into mature virus was almost as large as the quantity of viral protein which did appear in mature virus. The “early” and “late” proteins could be shown to have separate and distinct immunological properties. The role of this large quantity of “early” protein is discussed.     

Integrated proviral human immunodeficiency virus type 1 is present in CD4+ peripheral blood lymphocytes in healthy seropositive individuals.

Evidence of a latent human immunodeficiency virus type 1 (HIV-1) infection in healthy, seropositive individuals who do not have viral antigens in their sera and from whom virions cannot be rescued in cocultivation experiments was examined. Proviral DNA was detected by amplification by the polymerase chain reaction procedure. In each of 10 seropositive individuals, the presence of HIV-1 proviral sequences was demonstrated in their peripheral blood mononuclear cells. By using fluorescence-activated cell sorting, we obtained highly enriched subpopulations of peripheral blood mononuclear cells and found that the CD4+ T-cell subset is the cell subset that consistently harbors the HIV-1 proviral sequences. The number of HIV-1-infected CD4+ T cells was variable among the 10 healthy individuals, ranging from 1 in 100 to 1 in 40,000. While in vitro infection of CD4+ T cells causes down regulation and eventual loss of CD4 surface molecules, this is not true in vivo where it is only the CD4+ population that harbors the virus. This disparity may reflect differences between a latent infection in vivo with the lytic response of cells infected in vitro.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.