Significance of mirex-caused hypoglycemia and hyperlipidemia in rats

Treatment of rats with mirex (40 ppm in diet) caused hypoglycemia, liver enlargement, and inhibition of adrenal corticosteroid-synthesizing enzyme activity. At toxic dosages (20,000 ppm mirex in diet, which has a lethal toxicity-50 [LT-50] of ten days) poisoned female rats showed severe hypoglycemia, fatty liver, adrenal hyperplasia, hypophagia, lipid mobilization, and body weight (bw) loss. A 50 μg/kg intraperitoneal (IP) dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in male rats caused similar effects two days posttreatment. Hypoglycemia could be overcome by prednisone (which also inhibited adrenocorticoid-synthesizing enzyme activities) but not by streptozotocin treatment, indicating that hypoglycemia may be related to glucocorticoid deficiency resulting from inhibition of their synthesis and not by direct effects on pancreatic β-cells. Glucocorticoid deficiency could also cause increased release of adrenocorticoid hormone (ACTH), which may enhance fat mobilization caused by hypophagia.     

Effect of streptolysin S on liposomes Influence of membrane lipid composition on toxin action

The effect of the bacterial cytolytic toxin, streptolysin S, on liposomes composed of various phospholipids was investigated. Large unilamellar vesicles containing [¹⁴C]sucrose were prepared by reverse-phase evaporation, and membrane damage produced by the toxin was measured by following the release of labeled marker. The net charge of the liposomes had little or no effect on their susceptibility to steptolysin S and the toxin was about equally effective on liposomes composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylglycerol. Experiments with liposomes composed of synthetic phospholipids showed that the ability of the toxin to produce membrane damage depended on the degree of unsaturation of the fatty acyl chains. The order of sensitivity was C18 : 2 phosphatidylcholine > C18 : 1 phosphatidylcholine > C18 : 0 phosphatidylcholine = C16 : 0 phosphatidylcholine. Liposomes containing the latter two phospholipids were virtually unaffected by streptolysin S, and experiments with C18 : 0 phosphatidylcholine suggested that toxin activity does not bind to liposomes composed of phospholipids with saturated fatty acyl chains. The inclusion of 40 mol% cholesterol in C16 : 0 phosphatidylcholine and C18 : 0 phosphatidylcholine liposomes made these vesicles sensitive to streptolysin S. Egg phosphatidylcholine liposomes, which were unaffected at 0°C and 4°C became susceptible to the toxin at these temperatures when cholesterol was included. Liposomes composed of C14 : 0 phosphatidylcholine were unaffected by streptolysin S at temperatures below the chain-melting transition temperature (23°C) of this phospholipid, but became increasingly susceptible above this temperature. The results suggest that the fluidity of the phospholipid hydrocarbon chains in the membrane is important in streptolysin S action.     

Regulation of bifunctional proteins in cells: Lessons from the phospholipase Cβ/G protein pathway

Some proteins can serve multiple functions depending on different cellular conditions. An example of a bifunctional protein is inositide‐specific mammalian phospholipase Cβ (PLCβ). PLCβ is activated by G proteins in response to hormones and neurotransmitters to increase intracellular calcium. Recently, alternate cellular function(s) of PLCβ have become uncovered. However, the conditions that allow these different functions to be operative are unclear. Like many mammalian proteins, PLCβ has a conserved catalytic core along with several regulatory domains. These domains modulate the intensity and duration of calcium signals in response to external sensory information, and allow this enzyme to inhibit protein translation in a noncatalytic manner. In this review, we first describe PLCβ's cellular functions and regulation of the switching between these functions, and then discuss the thermodynamic considerations that offer insight into how cells manage multiple and competitive associations allowing them to rapidly shift between functional states.     

Subcellular localization of the APOBEC3 proteins during mitosis and implications for genomic DNA deamination

Humans have seven APOBEC3 DNA cytosine deaminases. The activity of these enzymes allows them to restrict a variety of retroviruses and retrotransposons, but may also cause pro-mutagenic genomic uracil lesions. During interphase the APOBEC3 proteins have different subcellular localizations: cell-wide, cytoplasmic or nuclear. This implies that only a subset of APOBEC3s have contact with nuclear DNA. However, during mitosis, the nuclear envelope breaks down and cytoplasmic proteins may enter what was formerly a privileged zone. To address the hypothesis that all APOBEC3 proteins have access to genomic DNA, we analyzed the localization of the APOBEC3 proteins during mitosis. We show that APOBEC3A, APOBEC3C and APOBEC3H are excluded from condensed chromosomes, but become cell-wide during telophase. However, APOBEC3B, APOBEC3D, APOBEC3F and APOBEC3G are excluded from chromatin throughout mitosis. After mitosis, APOBEC3B becomes nuclear, and APOBEC3D, APOBEC3F and APOBEC3G become cytoplasmic. Both structural motifs as well as size may be factors in regulating chromatin exclusion. Deaminase activity was not dependent on cell cycle phase. We also analyzed APOBEC3-induced cell cycle perturbations as a measure of each enzyme’s capacity to inflict genomic DNA damage. AID, APOBEC3A and APOBEC3B altered the cell cycle profile, and, unexpectedly, APOBEC3D also caused changes. We conclude that several APOBEC3 family members have access to the nuclear compartment and can impede the cell cycle, most likely through DNA deamination and the ensuing DNA damage response. Such genomic damage may contribute to carcinogenesis, as demonstrated by AID in B cell cancers and, recently, APOBEC3B in breast cancers.     

Characterization of a novel parainfluenza virus,caviid parainfluenza virus 3, from laboratory guinea pigs (Cavia porcellus)

A novel Respirovirus was isolated from nasopharyngeal swab specimens from clinically normal laboratory guinea pigs, and was characterized and named caviid parainfluenza virus 3 (CavPIV-3). The CavPIV-3 is enveloped, is 100 to 300 nm in diameter, and has a characteristic 15-nm-diameter chevron-shaped virus ribonucleocapsid protein. Sequence analysis of the fusion glycoprotein of CavPIV-3 revealed it to be 94% identical to human and guinea pig parainfluenza 3 (PIV-3) viruses and 80% identical to bovine PIV-3. To determine whether CavPIV-3 causes clinical disease in laboratory guinea pigs and to compare the serologic response of guinea pigs to CavPIV-3 and to other paramyxoviruses, an infection study was performed, in which groups of guinea pigs were inoculated with CavPIV-3, Sendai virus, simian virus 5 (SV-5), murine pneumonia virus (PVM), or bovine PIV-3 virus. During the course of the study, guinea pigs were maintained in an infectious disease suite, housed in Micro-Isolator cages, and were only manipulated under a laminar flow hood. Clinical signs of disease were not observed in any of the paramyxovirus-inoculated guinea pigs during the eight-week course of the study, and histologic signs of disease were not evident at necropsy eight weeks after inoculation. Guinea pigs inoculated with CavPIV-3, Sendai virus, PVM, and bovine PIV-3 developed robust homologous or heterologous serologic responses. In contrast, guinea pigs inoculated with SV-5 developed modest or equivocal serologic responses, as assessed by use of an enzyme-linked immunosorbent assay. Further, use of the SV-5 enzyme-linked immunosorbent assay resulted in the highest degree of non-specific reactivity among all of the paramyxovirus assays. In summary, CavPIV-3 is a novel guinea pig Respirovirus that subclinically infects laboratory guinea pigs, resulting in a robust serologic response, but no observed clinical or histologic disease. The CavPIV-3 fusion glycoprotein gene sequence is available from GenBank as accession No. AF394241, and the CavPIV-3 virus is available from the American Type Culture Collection as accession No. DR-1547.     

Destabilization of Yeast Micro- and Minisatellite DNA Sequences by Mutations Affecting a Nuclease Involved in Okazaki Fragment Processing (rad27) and DNA Polymerase δ (pol3-t)

We examined the effects of mutations in the Saccharomyces cerevisiae RAD27 (encoding a nuclease involved in the processing of Okazaki fragments) and POL3 (encoding DNA polymerase δ) genes on the stability of a minisatellite sequence (20-bp repeats) and microsatellites (1- to 8-bp repeat units). Both the rad27 and pol3-t mutations destabilized both classes of repeats, although the types of tract alterations observed in the two mutant strains were different. The tract alterations observed in rad27 strains were primarily additions, and those observed in pol3-t strains were primarily deletions. Measurements of the rates of repetitive tract alterations in strains with both rad27 and pol3-t indicated that the stimulation of microsatellite instability by rad27 was reduced by the effects of the pol3-t mutation. We also found that rad27 and pol3-01 (an allele carrying a mutation in the “proofreading” exonuclease domain of DNA polymerase δ) mutations were synthetically lethal.All eukaryotic genomes thus far examined contain many simple repetitive DNA sequences, tracts of DNA with one or a small number of bases repeated multiple times (48). These repetitive regions can be classified as microsatellites (small repeat units in tandem arrays 10 to 60 bp in length) and minisatellites (larger repeat units in tandem arrays several hundred base pairs to several kilobase pairs in length). In this paper, arrays with repeat units 14 bp or less will be considered microsatellites and arrays with longer repeat units will be considered minisatellites.Previous studies show that simple repetitive sequences are unstable relative to “normal” DNA sequences, frequently undergoing additions or deletions of repeat units, in Escherichia coli (24), Saccharomyces cerevisiae (12), and mammals (59). This mutability has two important consequences. First, it results in polymorphic loci that are useful in genetic mapping and forensic studies (15, 59). Second, although these repetitive tracts are usually located outside of coding sequences, alterations in the lengths of microsatellites or minisatellites located within coding sequences can produce frameshift mutations or novel protein variants (20, 22, 26).From studies of the effects of various mutations on microsatellite stability in yeast and E. coli (40) and the analysis of mutational changes caused by DNA polymerase in vitro (21), it is likely that most alterations reflect DNA polymerase slippage events (47). These events involve the transient dissociation of the primer and template strands during the replication of a microsatellite (Fig. ​(Fig.1).1). If the strands reassociate to yield an unpaired repeat on the primer strand, the net result is an addition of repeats (following a second round of DNA replication). Unpaired repeats on the template strand would result in a deletion by the same mechanism. Open in a separate windowFIG. 1“Classical” model for the generation of microsatellite alterations by DNA polymerase slippage. Two single strands of a replicating DNA molecule are shown, with each repeat unit indicated by a rectangle. Arrows indicate the 3′ ends of the strand, and the top and bottom strands represent the elongating primer strand and the template strand, respectively. Step 1, the primer and template strand dissociate; step 2, the primer and template strands reassociate in a misaligned configuration, resulting in an unpaired repeat on either the template strand (left side) or primer strand (right side); step 3, DNA synthesis is completed. If the unpaired repeats are not excised by the DNA mismatch repair system, after the next round of DNA synthesis one DNA molecule will be shortened by one repeat (left side) or lengthened by one repeat (right side).A number of mutations have been shown to elevate microsatellite instability. In E. coli (24, 46), yeast (44, 45), and mammalian cells (27), mutations in genes affecting DNA mismatch repair dramatically elevate the instability of a dinucleotide microsatellite. The most likely explanation of this result is that the DNA mismatches (unpaired repeats) resulting from DNA polymerase slippage events are efficiently removed from the newly synthesized strand by the DNA mismatch repair system. Thus, in the absence of mismatch repair, tract instability is elevated. From genetic studies, it has been found that mismatch repair in yeast efficiently corrects DNA mismatches involving 1- to 14-base loops (the size of the repeat units in microsatellites) but fails to correct mismatches involving loops larger than 16 bases (the size of the repeat units in minisatellites) (3, 41, 53). An inefficient mechanism, not involving the classical DNA mismatch repair system, is capable of correcting large DNA loops formed during meiotic recombination (19).In addition to mutations affecting DNA mismatch repair, some mutations affecting DNA replication in yeast destabilize microsatellites. Yeast strains bearing a null mutation in the RAD27 (RTH1) gene have high levels of instability of the dinucleotide poly(GT) and the trinucleotide CAG, specifically elevating single-repeat insertions (18, 39). RAD27 encodes the homolog of the mammalian FEN-1 protein, a 5′-to-3′ exonuclease (10, 11, 33). This nuclease activity is required for removing the terminal ribonucleotide residue from the 5′ end of the Okazaki fragment (9, 14, 35, 54, 55, 57); this step is necessary for the two adjoining fragments to be ligated together. FEN-1 appears to be active as either an exonuclease in the presence of a single-stranded gap upstream of the 5′ terminus or an endonuclease on a 5′ flap structure (13, 34). Since yeast strains that contain a null mutation in RAD27 grow poorly but are viable (38, 43), it is likely that less efficient nuclease activities that are also capable of 5′ Okazaki fragment processing are present in yeast. In addition to destabilizing dinucleotide microsatellites, rad27 strains have high levels of spontaneous mitotic recombination, elevated rates of forward mutation, and increased sensitivity to the alkylating agent methyl methanesulfonate (MMS) (18, 38, 43). In contrast to the mutations normally seen in mismatch repair mutants, i.e., point mutations or small frameshifts, the types of mutations observed in the absence of Rad27p are duplications of sequences flanked by short direct repeats (4 to 7 bp in length) (49). These duplications were not affected by the DNA mismatch repair system.The same class of sequences that are duplicated in the rad27 strains show an elevated rate (up to 1,000-fold) of deletion in strains containing a temperature-sensitive allele (pol3-t) of the yeast gene encoding DNA polymerase δ (52, 53). This mutant (initially named tex1) was isolated in a strain that exhibited an increased excision rate of a bacterial transposon with long terminal repeats inserted within a yeast gene (7). The pol3-t allele, which encodes a mutation (Gly₆₄₁ to Ala₆₄₁) (51) located near the putative nucleotide binding and active-site domains of the enzyme (58), is thought to diminish the rate of lagging-strand synthesis resulting in long stretches of single-stranded DNA on the lagging-strand template (8). This single-stranded DNA may have the potential to form intrastrand base-paired structures, creating interactions between short direct repeats. These interactions would result in an increased frequency of deletions caused by DNA polymerase slippage.Since rad27 and pol3-t mutations elevate the rates of duplications and deletions associated with short separated repeats in nonrepetitive DNA sequences, Kunkel et al. (22) suggested that these mutations could also destabilize minisatellites. In this paper, we examine the effects of rad27 and pol3-t mutations on the stability of simple repeats in which the repeat unit length varies between 1 and 20 bp. Our results show that both mutations destabilize both microsatellites and minisatellites, but that the mechanisms involved in the destabilization are different for the two mutations.     

Evolution of Influenza B Virus in Kuala Lumpur,Malaysia, between 1995 and 2008

Influenza B virus causes significant disease but remains understudied in tropical regions. We sequenced 72 influenza B viruses collected in Kuala Lumpur, Malaysia, from 1995 to 2008. The predominant circulating lineage (Victoria or Yamagata) changed every 1 to 3 years, and these shifts were associated with increased incidence of influenza B. We also found poor lineage matches with recommended influenza virus vaccine strains. While most influenza B virus lineages in Malaysia were short-lived, one circulated for 3 to 4 years.     

Nonmuscle Myosin-Dependent Synthesis of Type I Collagen

Type I collagen, synthesized in all tissues as the heterotrimer of two α1(I) polypeptides and one α2(I) polypeptide, is the most abundant protein in the human body. Here we show that intact nonmuscle myosin filaments are required for the synthesis of heterotrimeric type I collagen. Conserved 5′ stem-loop in collagen α1(I) and α2(I) mRNAs binds the RNA-binding protein LARP6. LARP6 interacts with nonmuscle myosin through its C-terminal domain and associates collagen mRNAs with the filaments. Dissociation of nonmuscle myosin filaments results in secretion of collagen α1(I) homotrimer, diminished intracellular colocalization of collagen α1(I) and α2(I) polypeptides (required for folding of the heterotrimer), and their increased intracellular degradation. Inhibition of the motor function of myosin has similar collagen-specific effects, while disruption of actin filaments has a general effect on protein secretion. Nonmuscle myosin copurifies with polysomes, and there is a subset of polysomes involved in myosin-dependent translation of collagen mRNAs. These results indicate that association of collagen mRNAs with nonmuscle myosin filaments is necessary to coordinately synthesize collagen α1(I) and α2(I) polypeptides. We postulate that LARP6/myosin-dependent mechanism regulates the synthesis of heterotrimeric type I collagen by coordinating the translation of collagen mRNAs.