Living nautilus embryos: preliminary observations.

Nautilus, long recognized as the most primitive living cephalopod, provides insight into molluscan evolution. Despite many attempts, embryos have not been observed until now. This report details the surface morphology and extraembryonic circulatory pattern. It was found that development, as in other extant cephalopods, is direct, without larval stages. There appears to be no embryonic protoconch associated with shell ontogeny.

Selective inhibition of selenocysteine tRNA maturation and selenoprotein synthesis in transgenic mice expressing isopentenyladenosine-deficient selenocysteine tRNA.

Selenocysteine (Sec) tRNA (tRNA([Ser]Sec)) serves as both the site of Sec biosynthesis and the adapter molecule for donation of this amino acid to protein. The consequences on selenoprotein biosynthesis of overexpressing either the wild type or a mutant tRNA([Ser]Sec) lacking the modified base, isopentenyladenosine, in its anticodon loop were examined by introducing multiple copies of the corresponding tRNA([Ser]Sec) genes into the mouse genome. Overexpression of wild-type tRNA([Ser]Sec) did not affect selenoprotein synthesis. In contrast, the levels of numerous selenoproteins decreased in mice expressing isopentenyladenosine-deficient (i(6)A(-)) tRNA([Ser]Sec) in a protein- and tissue-specific manner. Cytosolic glutathione peroxidase and mitochondrial thioredoxin reductase 3 were the most and least affected selenoproteins, while selenoprotein expression was most and least affected in the liver and testes, respectively. The defect in selenoprotein expression occurred at translation, since selenoprotein mRNA levels were largely unaffected. Analysis of the tRNA([Ser]Sec) population showed that expression of i(6)A(-) tRNA([Ser]Sec) altered the distribution of the two major isoforms, whereby the maturation of tRNA([Ser]Sec) by methylation of the nucleoside in the wobble position was repressed. The data suggest that the levels of i(6)A(-) tRNA([Ser]Sec) and wild-type tRNA([Ser]Sec) are regulated independently and that the amount of wild-type tRNA([Ser]Sec) is determined, at least in part, by a feedback mechanism governed by the level of the tRNA([Ser]Sec) population. This study marks the first example of transgenic mice engineered to contain functional tRNA transgenes and suggests that i(6)A(-) tRNA([Ser]Sec) transgenic mice will be useful in assessing the biological roles of selenoproteins.

Flavopiridol induces G1 arrest with inhibition of cyclin-dependent kinase (CDK) 2 and CDK4 in human breast carcinoma cells.

Flavopiridol (L86-8275), a N-methylpiperidinyl, chlorophenyl flavone, can inhibit cell cycle progression in either G1 or G2 and is a potent cyclin-dependent kinase (CDK) 1 inhibitor. In this study, we used MCF-7 breast carcinoma cells that are wild type for p53 and pRb positive and contain CDK4-cyclin D1 and MDA-MB-468 breast carcinoma cells that are mutant p53, pRb negative, and lack CDK4-cyclin D1 to investigate the G1 arrest produced by Flavopiridol. Recombinant CDK4-cyclin D1 was inhibited potently by Flavopiridol (Kiapp, 65 nM), competitive with respect to ATP. Surprisingly, CDK4 immunoprecipitates derived from Flavopiridol-treated MCF-7 cells (3 h, 300 nM Flavonolpiridol) had an approximately 3-fold increased kinase activity compared with untreated cells. Cyclin D and CDK4 levels were not different at 3 hr, but cyclin D levels and CDK4 kinase activity decreased thereafter. The phosphorylation state of pRb was shifted from hypercoincident to hypocoincident with the development of G1 arrest. Asynchronous MDA-MB-468 cells were inhibited in cell cycle progression at both G1 and G2 by Flavopiridol. Flavopiridol inhibited the in vitro kinase activity of CDK2 using an immune complex kinase assay (IC50, 100 nM at 400 microM ATP). Immunoprecipitated CDK2 kinase activity from either MCF-7 or MDA-MB-468 cells exposed to Flavopiridol (300 nM) for increasing time showed an initial increased activity (approximately 1.5-fold at 3 h) compared with untreated cells, followed by a loss of kinase activity to immeasurable levels by 24 h. This increased immunoprecipitated kinase activity was dependent on the Flavopiridol concentration added to intact cells and was associated with a reduction of CDK2 tyrosine phosphorylation. Cyclin E and A levels were not altered to the same extent as cyclin D, and neither CDK4 nor CDK2 levels were changed in response to Flavopiridol. Inhibition of the CDK4 and/or CDK2 kinase activity by Flavopiridol can therefore account for the G1 arrest observed after exposure to Flavopiridol.

Structural organization and expression of the gaegurin 4 gene of Rana rugosa.

Gaegurin 4 (GGN4) is a member of the antimicrobial peptide subfamily isolated from the skin of Rana rugosa. We cloned gDNA encoding GGN4 to study its gene organization and regulation of expression. The GGN4 gene occurs in single copy in the R. rugosa genome and contains a single intron of about 3.4 kb. The transcription start site is located 68 bases upstream of the translation initiation codon. The GGN4 gene was expressed both in Xenopus kidney epithelial cells (A6) and in Xenopus oocytes using the chloramphenicol acetyltransferase reporter gene system. The 5' flanking region of the GGN4 gene contains a dl binding site that is known to regulate acute phase immune response related gene expression in mammals and insects. The dl protein bound specifically to the GGN4 gene promoter region. Mutants that serially delete the 5' flanking region show that removal of the dl binding site inhibited GGN4 gene expression in both A6 cells and Xenopus oocytes. From these results, we propose that expression of the GGN4 gene may be regulated by the region containing the dl element which plays a key role in the regulation of antimicrobial peptide genes in Drosophila and mammals.

Replenishment of selenium deficient rats with selenium results in redistribution of the selenocysteine tRNA population in a tissue specific manner.

We reported previously that the selenium status of rats influences both the steady-state levels and distributions of two selenocysteine tRNA isoacceptors and that these isoacceptors differ by a single methyl group attached to the ribosyl moiety at position 34. In this study, we demonstrate that repletion of selenium-deficient rats results in a gradual, tissue-dependent shift in the distribution of these isoacceptors. Rats fed a selenium-deficient diet possess a greater abundance of the species unmethylated on the ribosyl moiety at position 34 compared to the form methylated at this position. A redistribution of the Sec-tRNA isoacceptors occurred in tissues of selenium-supplemented rats whereby the unmethylated form gradually shifted toward the methylated form. This was true in each of four tissues examined, muscle, kidney, liver and heart, although the rate of redistribution was tissue-specific. Muscle manifested a predominance of two minor serine isoacceptors under conditions of extreme selenium-deficiency which also appeared to respond to selenium. Ribosomal binding studies revealed that one of the two additional isoacceptors decodes the serine codeword, AGU, and the second decodes the serine codeword, UCU. Interestingly, muscle and heart were the slower tissues to return to a 'selenium adequate' tRNA distribution pattern.

Analysis of selenocysteine (Sec) tRNA([Ser]Sec) genes in Chinese hamsters.

Several recent observations have indicated that the primary structure of the Chinese hamster selenocysteine tRNA([Ser]sec) is different than those of other mammalian species. These reports prompted us to investigate the gene sequence for this tRNA in Chinese hamsters. Southern blotting of Chinese hamster ovary (CHO) genomic DNA derived from cultured cells with a tRNA([Ser]sec) probe indicated several hybridizing bands, and each of the corresponding genetic loci was isolated from a recombinant CHO library by molecular cloning. Sequence analysis of these regions indicated three likely pseudogenes and a single functional gene whose sequence differed from those of other mammals. Of these, only one pseudogene and the putative functional gene are actively transcribed following their microinjection into Xenopus oocytes. The possibility that the functional CHO tRNA([Ser]sec) evolved from an edited transcript is discussed.

The zebrafish genome contains two distinct selenocysteine tRNA[Ser]sec genes.

The zebrafish is widely used as a model system for studying mammalian developmental genetics and more recently, as a model system for carcinogenesis. Since there is mounting evidence that selenium can prevent cancer in mammals, including humans, we characterized the selenocysteine tRNA[Ser]sec gene and its product in zebrafish. Two genes for this tRNA were isolated and sequenced and were found to map at different loci within the zebrafish genome. The encoding sequences of both are identical and their flanking sequences are highly homologous for several hundred bases in both directions. The two genes likely arose from gene duplication which is a common phenomenon among many genes in this species. In addition, zebrafish tRNA[Ser]sec was isolated from the total tRNA population and shown to decode UGA in a ribosomal binding assay.

Multiple levels of regulation of selenoprotein biosynthesis revealed from the analysis of human glioma cell lines.

To gain a better understanding of the biological consequences of the exposure of tumor cells to selenium, we evaluated the selenium-dependent responses of two selenoproteins (glutathione peroxidase and the recently characterized 15-kDa selenoprotein) in three human glioma cell lines. Protein levels, mRNA levels, and the relative distribution of the two selenocysteine tRNA isoacceptors (designated mcm(5)U and mcm(5)Um) were determined for standard as well as selenium-supplemented conditions. The human malignant glioma cell lines D54, U251, and U87 were maintained in normal or selenium-supplemented (30 nM sodium selenite) conditions. Northern blot analysis demonstrated only minor increases in steady-state GSHPx-1 mRNA in response to selenium addition. Baseline glutathione peroxidase activity was 10.7 +/- 0.7, 7.6 +/- 0.7, and 4.3 +/- 0.7 nmol NADPH oxidized/min/mg protein for D54, U251, and U87, respectively, as determined by the standard coupled spectrophotometric assay. Glutathione peroxidase activity increased in a cell line-specific manner to 19.7 +/- 1.4, 15.6 +/- 2.1, and 6. 7 +/- 0.5 nmol NADPH oxidized/min/mg protein, respectively, as did a proportional increase in cellular resistance to H(2)O(2), in response to added selenium. The 15-kDa selenoprotein mRNA levels likewise remained constant despite selenium supplementation. The selenium-dependent change in distribution between the two selenocysteine tRNA isoacceptors also occurred in a cell line-specific manner. The percentage of the methylated isoacceptor, mcm(5)Um, changed from 35.5 to 47.2 for D54, from 38.1 to 47.3 for U251, and from 49.0 to 47.6 for U87. These data represent the first time that selenium-dependent changes in selenoprotein mRNA and protein levels, as well as selenocysteine tRNA distribution, were examined in human glioma cell lines.

Yeast asparagine (Asn) tRNA without Q base promotes eukaryotic frameshifting more efficiently than mammalian Asn tRNAs with or without Q base.

In this study, we compare the efficiency of Asn tRNA from mammalian sources with and without the highly modified queuosine (Q) base in the wobble position of its anticodon and Asn tRNA from yeast, which naturally lacks Q base, to promote frameshifting. Interestingly, no differences in the ability of the two mammalian Asn tRNAs to promote frameshifting were observed, while yeast tRNA(ASn)(-Q) promoted frameshifting more efficiently than its mammalian counterparts in both rabbit reticulocyte lysates and wheat germ extracts. The shiftability of yeast Asn tRNA is therefore not due, or at least not completely, to the lack of Q base and most likely the shiftiness resides in structural differences elsewhere in the molecule. However, we cannot absolutely rule out a role of Q base in frameshifting as wheat germ extracts and a lysate depleted of most of its tRNA and supplemented with calf liver tRNA contain both Asn tRNA with or without Q base.

Rapidly progressive dementia caused by nonenhancing primary lymphoma of the central nervous system.

A 76-year-old woman had rapidly progressive dementia over 4 months. Proton density- and T2-weighted MR images of the head showed increased signal in the periventricular and subcortical white matter of both cerebral hemispheres and the brain stem. Enhancement was not seen after administration of contrast material. Because of the rapid progression of the dementia and increasing signal abnormalities within the cerebral white matter, the patient underwent a craniotomy with biopsies of the right frontal lobe. Pathologic specimens were positive for malignant large-cell lymphoma, B-cell phenotype. The MR appearance is atypical for primary lymphoma of the central nervous system.

Selenocysteine incorporation directed from the 3'UTR: characterization of eukaryotic EFsec and mechanistic implications.

The mechanism of selenocysteine incorporation in eukaryotes has been assumed for almost a decade to be inherently different from that in prokaryotes, due to differences in the architecture of selenoprotein mRNAs in the two kingdoms. After extensive efforts in a number of laboratories spanning the same time frame, some of the essential differences between these mechanisms are finally being revealed, through identification of the factors catalyzing cotranslational selenocysteine insertion in eukaryotes. A single factor in prokaryotes recognizes both the selenoprotein mRNA, via sequences in the coding region, and the unique selenocysteyl-tRNA, via both its secondary structure and amino acid. The corresponding functions in eukaryotes are conferred by two distinct but interacting factors, one recognizing the mRNA, via structures in the 3' untranslated region, and the second recognizing the tRNA. Now, with these factors in hand, crucial questions about the mechanistic details and efficiency of this intriguing process can begin to be addressed.

Bacterial and polymorphonuclear leukocyte contribution to middle ear inflammation in chronic otitis media with effusion.

Bacteria can be cultured from approximately one third of chronic middle ear effusions, yet the contribution of these bacteria to the pathogenesis of chronic otitis media with effusion (OME) is not clear due to the absence of signs and symptoms of acute infection in most children with this disease. To explore the role of bacteria in chronic OME, lysozyme, lactoferrin, serum complement factors C3 and C5a, and polymorphonuclear leukocyte (PMNL) chemotaxin content was measured in 21 chronic middle ear effusion samples. Concentrations of lysozyme, lactoferrin, and chemotaxin were significantly higher in culture-positive than in sterile effusions. Lysozyme appeared to be contributed by both PMNL and non-PMNL sources in the middle ear space. These non-PMNL sources, presumably middle ear epithelial cells, accounted for 50% to 80% of the lysozyme variation in middle ear effusion. Although C3 and C5a were present in effusion, chemotaxin content correlated poorly with the C3 and C5a content, suggesting that chemotaxins were derived from bacterial peptides rather than from complement activation products. These results suggest that bacteria contribute to chronic middle ear inflammation with effusion. The eradication of bacteria from chronic middle ear effusion might disrupt the host responses which maintain chronic OME.

Selenocysteine tRNAs as central components of selenoprotein biosynthesis in eukaryotes.

Selenocysteine (Sec) tRNAs serve as carrier molecules for the biosynthesis of Sec from serine and to donate Sec to protein in response to specific UGA codons. In this study, we describe the current status of Sec tRNAs in higher animals and further we examine: (i) the Sec tRNA population in Drosophila; (ii) transcription of the Sec tRNA in vivo (in Xenopus oocytes) and in vitro (in Xenopus oocyte extracts); (iii) the effect of selenium on the Sec tRNA population in various rat tissues following replenishment of extremely selenium deficient rats with this element; and (iv) the biosynthesis of the modified bases on Sec tRNA in Xenopus oocytes.

Using surveys for management and measurement of health in developing countries.

National household surveys have been a basic statistical feature for many decades in the industrialized countries and more recently in the developing world. This paper deals with the potential of national household surveys for obtaining health information in developing countries. In this regard the United Nations National Household Survey Capability Programme (NHSCP) aims at collaborating with developing countries to establish a continuing flow of integrated statistics.

Complications associated with laser surgery.

All lasers are classified according to their potential for injury. Because medical lasers fall into the category having the greatest hazard potential, appropriate safeguards must be planned and undertaken. This article addresses the complications associated with the photoablative and thermal effects of lasers. Despite the laser's potential for injury, however, the probability of serious damage is negligible if the modality is used intelligently by the informed physician.

Lasers in podiatry and orthopedics.

Laser use in podiatry has proved very beneficial for many conditions of the foot. Laser use in orthopedics is beginning to evolve as the benefits that can be derived from the this technology are being realized. The CO2, Nd:YAG, and argon systems are being used and investigated, and new applications are being introduced.

High-throughput small molecule screening reveals structurally diverse compounds that inhibit the growth of Escherichia coli O157:H7 in vitro.

Escherichia coli O157:H7 colonizes the gastrointestinal tract of ruminants asymptomatically and may enter the human food supply through fecal contamination. A fraction of individuals infected by E. coli O157:H7 develop hemolytic uremic syndrome, a life-threatening condition. When individuals infected by E. coli O157:H7 are treated with certain antibiotics, an increased incidence of hemolytic uremic syndrome may result. This finding supports the need to identify novel compounds that can either reduce the load of E. coli O157:H7 entering the human food supply or serve as alternative therapeutic treatments for infected individuals. We developed a high-throughput turbidometric assay to identify novel compounds that inhibit E. coli O157:H7 growth. Pin transfers were performed to introduce small molecule libraries into 384-well plates, where each well contained approximately 5.0 log CFU of E. coli O157:H7. Plates were incubated at 37°C for 18 h, and the optical density was measured to determine the effect of each small molecule. A total of 64,562 compounds were screened in duplicate, and 43 unique compounds inhibited E. coli O157:H7 growth. Thirty-eight of the 43 inhibitory compounds belonged to known bioactive libraries, and the other 5 compounds were from commercial libraries derived from splitting and pooling. Inhibitory compounds from known bioactive libraries were most frequently therapeutic antibiotics (n = 34) but also included an antiviral compound, a compound that disrupts the citric acid cycle, and two biguanide compounds, which have been used for various nonclinical applications. We identified two novel compounds (i.e., biguanides) that should be studied further for their ability to reduce pathogen populations in foods.

Naturally colonized beef cattle populations fed combinations of yeast culture and an ionophore in finishing diets containing dried distiller's grains with solubles had similar fecal shedding of Escherichia coli O157:H7.

Beef steers (n = 252) were used to evaluate the effects of dietary supplement on fecal shedding of Escherichia coli O157:H7. Seven pens of 9 steers (63 steers per treatment) were fed diets supplemented with or without yeast culture (YC) or monensin (MON) and their combination (YC × MON). YC and MON were offered at 2.8 g/kg and 33 mg/kg of dry matter intake, respectively. Environmental sponge samples (from each pen floor, feed bunk, and water trough) were collected on day 0. Rectal fecal grab samples were collected on days 0, 28, 56, 84, 110, and 125. Samples were collected and pooled by pen and analyzed for presumptive E. coli O157:H7 colonies, which were confirmed by a multiplex PCR assay and characterized by pulsed-field gel electrophoresis (PFGE) typing. On day 0, E. coli O157:H7 was detected in 7.0% of feed bunk samples and 14.3% of pen floor samples but in none of the water trough samples. The 71.4% prevalence of E. coli O157:H7 in fecal samples on day 0 decreased significantly (P < 0.05) over time. E. coli O157:H7 fecal shedding was not associated with dietary treatment (P > 0.05); however, in cattle fed YC and YC × MON fecal shedding was 0% by day 28. Eight Xba I PFGE subtypes were identified, and a predominant subtype and three closely related subtypes (differing by three or fewer bands) accounted for 78.7% of environmental and fecal isolates characterized. Results from this study indicate that feeding YC to cattle may numerically decrease but not eliminate fecal shedding of E. coli O157:H7 at the onset of treatment and that certain E. coli O157 subtypes found in the feedlot environment may persist in feedlot cattle.

The effects of pre-slaughter pig management from the farm to the processing plant on pork quality.

Two experiments (Exp.1, n=80; Exp.2, n=144) were conducted to determine the effects of pre-slaughter pig management on pork quality by monitoring blood lactate concentration ([LAC]) during marketing. [LAC] was measured at: (1) baseline at farm, (2) post-loading on truck, (3) pre-unloading after transport, (4) post-unloading at plant, (5) post-lairage, (6) post-movement to stun, and (7) exsanguination. Pearson correlations were used to determine relationships between [LAC] and meat quality. Higher [LAC] post-loading or a greater change in [LAC] during loading resulted in increased 24h pH (P=0.002, P=0.0006, Exp.1; P=0.0001, P=0.01, Exp.2, respectively), decreased L* (P=0.03, P=0.04; P=0.001, P=0.01) and decreased drip loss (P=0.02, P=0.12; P=0.002, P=0.01). Even though improved handling during loading is important to animal well-being, it will not necessarily translate into improved pork quality.