Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis

Inconsistent with the view that hair follicle stem cells reside in the matrix area of the hair bulb, we found that label-retaining cells exist exclusively in the bulge area of the mouse hair follicle. The bulge consists of a subpopulation of outer root sheath cells located in the midportion of the follicle at the arrector pili muscle attachment site. Keratinocytes in the bulge area are relatively undifferentiated ultrastructurally. They are normally slow cycling, but can be stimulated to proliferate transiently by TPA. Located in a well-protected and nourished environment, these cells mark the lower end of the "permanent" portion of the follicle. Our findings, plus a reevaluation of the literature, suggest that follicular stem cells reside in the bulge region, instead of the lower bulb. This new view provides insights into hair cycle control and the possible involvement of hair follicle stem cells in skin carcinogenesis.     

Binding of a distamycin-ellipticine hybrid molecule to DNA and chromatin: spectroscopic, biochemical, and molecular modeling investigations.

A bifunctional molecule in which an ellipticine chromophore is attached to a distamycin residue via a diaminopropyl tether has been designed and synthesized in the expectation of creating a hybrid molecule capable of bidentate binding to DNA by both intercalation and minor-groove interactions. The strength and mode of binding to DNA of this conjugate have been studied by means of circular and linear dichroism as well as by stopped-flow kinetics and measurements of reactivity toward a chemical probe. The results converge to reveal that the ellipticine moiety of the hybrid largely dominates the binding reaction with DNA. In the presence of chromatin, the hybrid molecule binds preferentially to the internucleosomal DNA, a preference dictated by its intercalating chromophore. Theoretical computations were performed on the comparative complexation energies of distamycin, the ellipticine derivative, and the hybrid ligand with a B-representative octanucleotide, d(GCATATGC)2. The best binding configuration of the ellipticine derivative locates its aminoalkyl side chain in the minor groove where distamycin is also present. The molecular modeling analysis fully supports the involvement of a bimodal binding process for the hybrid and reveals that the binding of the conjugate to DNA favors a pronounced bending toward the minor groove. This effect is attributed to intercalation of the ellipticine chromophore. An interesting link is established between the DEPC reactivity experiments and the theoretical computations, suggesting that DEPC can be used as a probe for drug-induced DNA bending. On the basis of these results, we propose the design of a new hybrid ligand bearing an additional positively-charged amidine side chain to confer higher DNA-binding affinity.     

Initiation of minus-strand RNA synthesis by the brome mosaicvirus RNA-dependent RNA polymerase: use of oligoribonucleotide primers.

Various DNA- and RNA-dependent RNA polymerases have been reported to use oligoribonucleotide primers to initiate nucleic acid synthesis. For the brome mosaic virus RNA-dependent RNA polymerase (RdRp), we determined that in reactions performed with limited GTP concentrations, minus-strand RNA synthesis can be stimulated by the inclusion of guanosine monophosphate or specific oligoribonucleotides. Furthermore, guanylyl-3',5'-guanosine (GpG) was incorporated into minus-strand RNA and increased the rate of minus-strand RNA synthesis. In the presence of GpG, RdRp's Km for GTP decreased from 50 microM to approximately 3 microM while the Kms for other nucleotides were unaffected. These results have implications for the mechanism of initiation by RdRp.     

Resuscitation of Vibrio cholerae O1 strain TSI-4 from a viable but nonculturable state by heat shock

Abstract Vibrio cholerae strain TSI-4 was incubated in an M9 salt solution at 15 °C for more than 100 days. The plate counts showed no viable cells on day 30, but a broth culture from that day showed the growth of bacteria. However, after 35 days the bacteria entered the nonculturable state, based on the assessment of both the plate counts and broth culture. A portion of the culture was heated at 45 °C for 1 min in a water bath and subsequently plated onto a nutrient agar plate. More than 1000 colonies were recovered after this heat-shock treatment. The recovered cells showed the same chromosomal DNA pattern in the restriction map and the same outer membrane protein pattern in SDS-PAGE. Recovery of viable cells by heat-shock was achieved in cultures grown on M9 salt but not from cultures grown in phosphate-buffered saline. This suggests that the presence of NH₄Cl in the M9 salt solution may support the growth of the bacteria in a low nutrient medium, while also playing an important role in resuscitation.     

Analysis of Lactobacillus phages and bacteriocins in American dairy products and characterization of a phage isolated from yogurt.

Yogurt and acidophilus milk that contain Lactobacillus acidophilus could promote human health because L. acidophilus can inhibit enteric and food-borne microbial pathogens. To evaluate the stability of diary L. acidophilus cultures, we studied whether some diary lactobacilli could be inhibited by phages or bacteriocins released by other dairy lactobacilli. From 20 yogurts and two acidophilus milks purchased at local food markets, 38 Lactobacillus strains were isolated. Eight Lactobacillus type strains were used as controls. With mitomycin induction and agar spot assay, phages and bacteriocins were isolated from these strains and their activities were analyzed. Lactobacillus strains from 11 yogurts released phages, while the strains from most of the remaining products released bacteriocins. One phage, designated phi y8, was characterized. It was spontaneously released from its host strain L. acidophilus Y8, at a rate of about 10(4)/ml. This phage lysed nine other dairy Lactobacillus strains tested. It had a burst size of 100, an elongated prolate head of 39 by 130 nm, a long, flexible but noncontractile tail of 300 nm, and a 54.3-kb linear double-stranded DNA. DNA fingerprinting analysis indicated that L. acidophilus phages of nine yogurts in this study belonged to the same type as phi y8. Although they may be sensitive to bacteriocins, all lysogens resisted further phage attacks, whereas most nonlysogens were sensitive to both phages and bacteriocins. Therefore, Lacotbacillus cultures of some American yogurts and acidophilus milks may be unstable or unsafe because they can either be inhibited by phages or bacteriocins or release them to inhibit lactobacilli or other diary products.     

A single tryptophan on M2 of glutamate receptor channels confers high permeability to divalent cations.

Ionotropic glutamate receptors (iGluRs) of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate subtype display lower permeability to Ca2+ than the N-methyl-D-aspartate (NMDA) subtype. The well-documented N/Q/R site on the M2 transmembrane segment (M2) is an important determinant of the distinct Ca2+ permeability exhibited by members of the non-NMDA receptor subfamily. This site, however, does not completely account for the different permeation properties displayed by non-NMDA and NMDA receptors, suggesting the involvement of other molecular determinants. We have identified additional molecular elements on M2 of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptor GluR1 that specify its permeation properties. Higher permeability to divalent over monovalent cations is conferred on GluR1 by a tryptophan at position 577, whereas blockade by external divalent cations is imparted by an asparagine at position 582. Hence, the permeation properties of ionotropic glutamate receptors appear to be primarily specified by two distinct determinants on M2, the well-known N/Q/R site and the newly identified L/W site. These findings substantiate the notion that M2 is a structural component of the pore lining.     

Is there an error correcting code in the base sequence in DNA?

Modern methods of encoding information into digital form include error check digits that are functions of the other information digits. When digital information is transmitted, the values of the error check digits can be computed from the information digits to determine whether the information has been received accurately. These error correcting codes make it possible to detect and correct common errors in transmission. The sequence of bases in DNA is also a digital code consisting of four symbols: A, C, G, and T. Does DNA also contain an error correcting code? Such a code would allow repair enzymes to protect the fidelity of nonreplicating DNA and increase the accuracy of replication. If a linear block error correcting code is present in DNA then some bases would be a linear function of the other bases in each set of bases. We developed an efficient procedure to determine whether such an error correcting code is present in the base sequence. We illustrate the use of this procedure by using it to analyze the lac operon and the gene for cytochrome c. These genes do not appear to contain such a simple error correcting code.     

Evidence for ion chain mechanism of the nonlinear charge transport of hydrophobic ions across lipid bilayers.

The conductivity across a lipid bilayer by tetraphenylborate anion is increased 10-fold on the photoformation of lipophilic porphyrin cations. The cations alone have negligible conductivity. This nonlinear photogenerated increase of ion conductivity is termed the photogating effect. Substitution of H by Cl in the para position of tetraphenylborate leads to a 100-fold enhancement of conductivity, whereas the dark conductivities for this and other substituted borates are the same. Moreover, the halo-substituted borates show a large enhancement of conductivity in the low concentration range (10(-8) M), whereas that of tetraphenylborate is small and space charge is negligible. The enhanced ion conductivity has great structural sensitivity to the structure of the anion, the cation, and the lipid, whereas the partition coefficient of all the borates and the concentration of photoformed cations are only slightly affected. The photogated ion transport has a twofold larger activation energy than transport in the dark. Time-resolved photocurrents and voltages demonstrate that the translocation rate of the porphyrin cation is also enhanced 100-fold by the Cl-borate anion but only 10-fold by the H-borate anion. For these reasons the nonlinear gating effect cannot be explained by electrostatics alone, but requires an ion chain or ion aggregate mechanism. Kinetic modeling of the photoinduced current with a mixed cation-anion ion chain can fit the data well. The photogating effect allows the direct study of ion interactions within the bilayer.     

Cloning and functional characterization of LCR-F1: a bZIP transcription factor that activates erythroid-specific,human globin gene expression.

DNase I hypersensitive site 2 (HS 2) of the human beta-globin Locus Control Region (LCR) directs high level expression of the beta-globin gene located 50 kilobases downstream. Experiments in cultured cells and in transgenic mice demonstrate that duplicated AP1-like sites in HS 2 are required for this powerful enhancer activity. A cDNA clone encoding a basic, leucine-zipper protein that binds to these sites was isolated and designated Locus Control Region-Factor 1 (LCR-F1). This protein is a member of a new family of regulatory factors that contain a 63 amino acid ''CNC domain'' overlapping the basic region. This domain is approximately 70% identical in the Drosophila Cap N Collar (CNC) protein, NF-E2 and LCR-F1. LCR-F1 transactivates an HS 2/gamma-globin reporter gene over 170-fold in transient transfection experiments specifically in erythroid cells. These results suggest that LCR-F1 may be a critical factor involved in LCR-mediated, human globin gene expression.