A study of connective tissue macromolecules in skin of mice with goldthioglucose-induced obesity.

The effect of obesity on the connective tissue composition of skin was investigated in mice with goldthioglucose (GTG)-induced obesity. Four months after GTG treatment, the obese animals were sacrificed. Acid mucopolysaccharides, glycoproteins, collagen, and elastin were analyzed in the skin and compared to the controls. Total MPS in the skin from obese animals decreased, reflected mostly in hyaluronic acid. Chondroitin showed an increase over controls. The content of soluble glycoproteins varied; total carbohydrate and sialic acid of the glycoprotein tended to increase with obesity. Collagen and elastin both tended to decrease with obesity.     

High-Resolution Functional Profiling of the Norovirus Genome

Human noroviruses (HuNoV) are a major cause of nonbacterial gastroenteritis worldwide, yet details of the life cycle and replication of HuNoV are relatively unknown due to the lack of an efficient cell culture system. Studies with murine norovirus (MNV), which can be propagated in permissive cells, have begun to probe different aspects of the norovirus life cycle; however, our understanding of the specific functions of the viral proteins lags far behind that of other RNA viruses. Genome-wide functional profiling by insertional mutagenesis can reveal protein domains essential for replication and can lead to generation of tagged viruses, which has not yet been achieved for noroviruses. Here, transposon-mediated insertional mutagenesis was used to create 5 libraries of mutagenized MNV infectious clones, each containing a 15-nucleotide sequence randomly inserted within a defined region of the genome. Infectious virus was recovered from each library and was subsequently passaged in cell culture to determine the effect of each insertion by insertion-specific fluorescent PCR profiling. Genome-wide profiling of over 2,000 insertions revealed essential protein domains and confirmed known functional motifs. As validation, several insertion sites were introduced into a wild-type clone, successfully allowing the recovery of infectious virus. Screening of a number of reporter proteins and epitope tags led to the generation of the first infectious epitope-tagged noroviruses carrying the FLAG epitope tag in either NS4 or VP2. Subsequent work confirmed that epitope-tagged fully infectious noroviruses may be of use in the dissection of the molecular interactions that occur within the viral replication complex.     

Genes for serum amyloid A proteins map to Chromosome 7 in the mouse

Summary Several restriction fragment length variants have been detected among inbred strains using a mouse serum amyloid A cDNA clone. Five variants were shown to segregate as a single genetic unit and were mapped to Chromosome 7 between the glucose phosphate isomerase locus (Gpi-1) and the pink eye dilution locus (p) using recombinant inbred and congenic strains. The finding that no major MspI or BclI restriction fragments were shared between digests of DNAs from a Chromosome 7 congenic strain and its inbred partner, indicate that most, and probably all, sequences detected with the probe are clustered on Chromosome 7. Aneuploid mapping was used to show that the serum amyloid A gene complex (Saa) is proximal to the Chromosome 7 breakpoint in T(7;X)1Ct, a translocation in which the middle third of Chromosome 7 is inserted into the X-chromosome. A survey of inbred strains revealed a single common Saa haplotype and eight rare haplotypes. The complex distribution of 14 different variants suggests that recombination may have played a role in haplotype evolution.This work was supported by grants GM18684 and CA33093 from the National Institute of General Medical Sciences and the National Cancer Institute, respectively.     

Paleomicrobiology: Revealing Fecal Microbiomes of Ancient Indigenous Cultures

Coprolites are fossilized feces that can be used to provide information on the composition of the intestinal microbiota and, as we show, possibly on diet. We analyzed human coprolites from the Huecoid and Saladoid cultures from a settlement on Vieques Island, Puerto Rico. While more is known about the Saladoid culture, it is believed that both societies co-existed on this island approximately from 5 to 1170 AD. By extracting DNA from the coprolites, followed by metagenomic characterization, we show that both cultures can be distinguished from each other on the basis of their bacterial and fungal gut microbiomes. In addition, we show that parasite loads were heavy and also culturally distinct. Huecoid coprolites were characterized by maize and Basidiomycetes sequences, suggesting that these were important components of their diet. Saladoid coprolite samples harbored sequences associated with fish parasites, suggesting that raw fish was a substantial component of their diet. The present study shows that ancient DNA is not entirely degraded in humid, tropical environments, and that dietary and/or host genetic differences in ancient populations may be reflected in the composition of their gut microbiome. This further supports the hypothesis that the two ancient cultures studied were distinct, and that they retained distinct technological/cultural differences during an extended period of close proximity and peaceful co-existence. The two populations seemed to form the later-day Taínos, the Amerindians present at the point of Columbian contact. Importantly, our data suggest that paleomicrobiomics can be a powerful tool to assess cultural differences between ancient populations.     

Amplified Rnase H Activity in Escherichia coli B/R Increases Sensitivity to Ultraviolet Radiation

Strains of E. coli B/r transformed with the plasmid pSK760 were found to be sensitized to inactivation by ultraviolet radiation (UV) and to have elevated levels of RNase H activity. Strains transformed with the carrier vector pBR322 or the plasmid pSK762C derived from pSK760 but with an inactivated rnh gene were not sensitized. UV-inactivation data for strains having known defects in DNA repair and transformed with pSK760 suggested an interference by RNase H of postreplication repair: uvrA cells were strongly sensitized, wild-type and uvrA recF cells were moderately sensitized and recA cells were not sensitized; and minimal medium recovery was no longer apparent in sensitized uvrA cells. Biochemical studies showed that post-UV DNA synthesis was sensitized and that the smaller amounts of DNA synthesized after irradiation, while of normal reduced size as indicated by sedimentation position in alkaline sucrose gradients, did not shift to a larger size (more rapidly sedimenting) upon additional incubation. We suggest an excess level of RNase H interferes with reinitiation of DNA synthesis on damaged templates to disturb the normal pattern of daughter strand gaps and thereby to inhibit postreplication repair.     

Interactions of ovalbumin and of its putative signal sequence with phospholipid monolayers. Possible importance of differing lateral stabilities in protein translocation.

Surface properties of ovalbumin and of its putative signal sequence, and their interactions with phospholipids at an air-water interface, have been studied. The mature protein can form an interfacial film spontaneously from its bulk solution, whereas the signal sequence cannot. Mature ovalbumin also penetrates phospholipid monolayers from the subphase (independently of the type of phospholipid present), whereas its signal sequence does not. The surface stability of a spread film of the signal sequence is, however, higher than that of a film of mature ovalbumin. Above specific threshold concentrations of signal peptide and of mature ovalbumin in mixed films with phospholipids, two separate phases are formed. In such immiscible films, the signal sequence peptide is also able to support a higher lateral surface pressure than mature ovalbumin, at corresponding areas of peptide and mature protein in the mixed monolayers. It is suggested that the differing lateral stabilities of ovalbumin and of its putative signal sequence may be relevant to the translocation of ovalbumin across the membrane of the endoplasmic reticulum, and a scheme for its translocation is proposed that is based on these properties.     

Analogue pigment studies of chromophore-protein interactions in metarhodopsins

Several analogue pigments have been prepared containing retinals altered at the cyclohexyl ring or proximal to the aldehyde group in order to examine the role of the chromophore in the formation of the metarhodopsin I and II states of visual pigments. Deletion of the 13-methyl group on the isoprenoid chain did not affect metarhodopsin formation. However, analogue pigments containing chromophores with modified rings did not show the typical absorption changes associated with the metarhodopsin transitions of native or regenerated rhodopsins. In particular, 4-hydroxyretinal pigments did not show clear transitions between the metarhodopsin I and metarhodopsin II states. Pigment formed with an acyclic retinal showed no evidence by absorption spectroscopy of metarhodopsin formation. A retinal altered by substitution of a five-membered ring containing a nitroxide required a more acidic pH than the native pigment for formation of the metarhodopsin II state. ESR data suggest that the ring remains buried within the protein through the metarhodopsin II state. However, the Schiff base linkage is susceptible to hydrolysis of hydroxylamine in the metarhodopsin II state. These data indicate that (1), in the transition from rhodopsin to metarhodopsin II, major protein conformational changes are occurring near the lysine-retinal linkage whereas the ring portion of the chromophore remains deeply buried within the protein and (2) pigment absorptions characteristic of the metarhodopsin I and II states may be due to specific protein-chromophore interactions near the region of the chromophore ring.     

Effect of seaweed concentrate on the growth and mineral nutrition of nutrient-stressed lettuce

The effects of the seaweed concentrate Kelpak on the growth and mineral nutrition of lettuce plants grown under conditions of varying nutrient supply were investigated. Kelpak significantly increased the yield and the concentration and amounts of Ca, K and Mg in the leaves of lettuce receiving an adequate supply of nutrients, but had little effect on nutrient stressed plants. Results are discussed in relation to the physiological mechanisms that have been proposed to explain the beneficial effects of seaweed concentrate on plants.author for correspondence