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The human type I hair keratin subfamily comprises nine individual members, which can be subdivided into three groups. Group A (hHa1, hHa3-I, hHa3-II, hHa4) and B (hHa7, hHa8) each contains structurally related hair keratins, whereas group C members hHa2, hHa5, and hHa6 represent structurally rather unrelated hair keratins. Antibodies produced against these individual hair keratins, first analyzed for specificity by one- dimensional Western blots of total hair keratins, were used to establish the two-dimensional catalog of the human type I hair keratin subfamily. The catalog comprises two different series of type I hair keratins: a strongly expressed, Coomassie-stainable series containing hair keratins hHa1, hHa3-I/II, hHa4, and hHa5, and a weakly expressed, immunodetectable series harboring hHa2, hHa6 hHa7, and hHa8. In situ hybridization and immunohistochemical expression studies on scalp follicles show that two hair keratins, hHa2 and hHa5, define the early stage of hair differentiation, i.e. hHa5 expression in hair matrix and hHa5/hHa2 coexpression in the early hair cuticle cells. Whereas cuticular differentiation proceeds without the expression of further type I hair keratins, matrix cells embark on the cortical pathway by sequentially expressing hHa1, hHa3-I/II, and hHa4, which are supplemented by hHa6 at an advanced stage of cortical differentiation, and hHa8, which is expressed heterogeneously in cortex cells. Thus, six type I hair keratins are involved in the terminal differentiation of anagen hairs. The expression of hHa7 is conspicuously different from that of the other hair keratins in that it does not occur in the large anagen follicles of terminal scalp hairs but only in central cortex cells of the rare and small follicle type that gives rise to vellus hairs.

Bmp2, a highly conserved member of the transforming growth factor-beta gene family, is crucial for normal development. Retinoic acid, combined with cAMP analogs, sharply induces the Bmp2 mRNA during the differentiation of F9 embryonal carcinoma cells into parietal endoderm. Retinoic acid (RA) also induces the Bmp2 gene in chick limb buds. Since normal Bmp2 expression may require an endogenous retinoid signal and aberrant Bmp2 expression may cause some aspects of RA-induced teratogenesis, we studied the mechanism underlying the induction of Bmp2. Measurements of the Bmp2 mRNA half-life and nuclear run-on assays indicated that RA stimulated the transcription rate of the Bmp2 gene. The results of ribonuclease protection and primer extension assays indicated that Bmp2 transcription started 2,127 nucleotides upstream of the translation start site in F9 cells. To identify genetic elements controlling this transcription rate increase, upstream and downstream genomic sequences flanking the Bmp2 gene were screened using chloramphenicol acetyltransferase reporter genes in F9 cells and beta-galactosidase reporter genes in Saccharomyces cerevisiae that were cotransformed with retinoic acid receptor and retinoid X receptor expression plasmids. RA-dependent transcriptional activation was detected between base pairs -2,373 and -2,316 relative to the translation start site. We also identified a required Sp1 binding site between -2,308 and -2,298. The data indicate that Bmp2 is directly regulated by retinoic acid-bound receptors and Sp1.

Sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) is critical for initiating differentiation of the PC12 cell to a sympathetic-like neurone. The neuropeptide, pituitary adenylyl cyclase-activating peptide (PACAP), has been demonstrated to cause cells to adopt a neuronal phenotype, although the mechanism of this activity is unclear. PACAP through its type I receptor stimulates a biphasic activation of ERK1/2; a >10-fold increase within 5 min, followed by a >5-fold increase that is sustained for >/=60 min. An equivalent stimulation is seen in PC12 cells expressing a dominant negative Ras mutant. However, the mitogen-activated kinase/ERK kinase 1/2 (MEK1/2) inhibitor PD98059 blocked both PACAP-induced stimulation of ERK1/2 activity and neurite outgrowth. Thus, the activation signal from the PACAP type I receptor on the ERK1/2 cascade pathway is received downstream of Ras, either at Raf or MEK. Down-regulation of protein kinase C or its inhibition by calphostin C blocked the ability of PACAP to stimulate ERK1/2. We conclude that activation of PACAP type I receptor activates protein kinase C, which then activates the ERK1/2 cascade in a Ras-independent manner at either Raf or MEK1/2.

Polymerase chain reaction-based screening of an arrayed human P1 artificial chromosome (PAC) library using primer pairs specific for the human type I hair keratins hHa3-II or hHa6, led to the isolation of two PAC clones, which covered 190 kilobase pairs (kbp) of genomic DNA and contained nine human type I hair keratin genes, one transcribed hair keratin pseudogene, as well as one orphan exon. The hair keratin genes are 4-7 kbp in size, exhibit intergenic distances of 5-8 kbp, and display the same direction of transcription. With one exception, all hair keratin genes are organized into 7 exons and 6 positionally conserved introns. On the basis of sequence homologies, the genes can be grouped into three subclusters of tandemly arranged genes. One subcluster harbors the highly related genes hHa1, hHa3-I, hHa3-II, and hHa4. A second subcluster of highly related genes comprises the novel genes hHa7 and hHa8, as well as pseudogene PsihHaA, while the structurally less related genes hHa6, hHa5, and hHa2 are constituents of the third subcluster. As shown by reverse transcription-polymerase chain reaction, all hair keratin genes, including the pseudogene, are expressed in the human hair follicle. The transcribed pseudogene PsihHaA contains a premature stop codon in exon 4 and exhibits aberrant pre-mRNA splicing. Evolutionary tree construction reveals an early divergence of hair keratin genes from cytokeratin genes, followed by the segregation of the genes into the three subclusters. We suspect that the 190-kbp domain contains the entire complement of human type I hair keratin genes.

While olfactory neurons of silk moths are well known for their exquisite sensitivity to sex pheromone odorants, molecular mechanisms underlying this sensitivity are poorly understood. In searching for proteins that might support olfactory mechanisms, we characterized the protein profile of olfactory neuron receptor membranes of the wild silk moth Antheraea polyphemus. We have purified and cloned a prominent 67-kDa protein which we have named Snmp-1 (sensory neuron membrane protein-1). Northern blot analysis suggests that Snmp-1 is uniquely expressed in antennal tissue; in situ hybridization and immunocytochemical analyses show that Snmp-1 is expressed in olfactory neurons and that the protein is localized to the cilia, dendrites, and somata but not the axons. Snmp-1 mRNA expression increases significantly 1-2 days before the end of adult development, coincident with the functional maturation of the olfactory system. Sequence analysis suggests Snmp-1 is homologous with the CD36 protein family, a phylogenetically diverse family of receptor-like membrane proteins. CD36 family proteins are characterized as having two transmembrane domains and interacting with proteinaceous ligands; Snmp-1 is the first member of this family identified in nervous tissue. These findings argue that Snmp-1 has an important role in olfaction; possible roles of Snmp-1 in odorant detection are discussed.

In this study, we have elucidated the molecular mechanisms underlying the expression of an acidic 41-kDa protein inherited as an autosomal dominant trait of the hair keratin pattern of about 5% of the human population. We show that this protein is a size variant of the large type I hair cortex keratin hHa1 due to a genetic polymorphism in the hHa1 gene. We detected a G-A substitution in the 5' splice site of intron 6 of the hHa1 gene, which segregates with the 41-kDa protein phenotype in two pedigrees and is responsible for the formation of an abnormally spliced hHa1 mRNA species. The use of an alternative 5' splice site leads to the retention of 41 nucleotides of the initial intron 6 sequences in the mature transcript. The open reading frame of the aberrant mRNA creates a premature stop codon immediately downstream of the mutation site. The resulting hHa1 protein variant, hHa1-t, is about 6-kDa smaller than the 47-kDa hHa1 hair keratin and lacks the complete nonhelical tail domain. We show that the tailless hHa1-t is functional, since both recombinant hHa1 and hHa1-t form identical keratin intermediate filaments when assembled in vitro with a type II hair keratin partner. This finding confirms the view of a noninvolvement of the keratin tail domain in filament assembly and explains the lack of a pathological hair phenotype in hHa1-t positive individuals.

The human malaria parasite Plasmodium falciparumhas two extrachromosomal DNAs associated with organelles whose function is unclear. Both genomes encode ribosomal RNAs (rRNAs) that are distinct from the nuclear-encoded rRNAs. Secondary structure analysis of all the P. falciparumrRNAs indicates that only the large subunit (LSU) rRNA encoded by the plastid-like genome is the target for thiostrepton. Indeed we find that thiostrepton inhibits growth of the parasite in the micromolar range which is 10-fold below concentrations with observable effects on total protein synthesis. We have further examined selective effects of thiostrepton on the plastid function by comparing differential effects of the drug on cytoplasmic and organellar encoded transcripts. Treatment with either thiostrepton or rifampin, an inhibitor of organellar and eubacterial RNA polymerase, both showed disappearance of organellar-encoded RNA transcripts within 6 h of treatment while transcripts of a nuclear-encoded mRNA remained constant for at least 8 h of treatment. Hence, we show a selective effect on organelle function that is suggestive of interference in the protein synthesis apparatus of the plastid. Sensitivity of P. falciparumto thiostrepton confirms that the plastid-like genome is essential for the erythrocytic cycle and presents a novel therapeutic site for this class of antibiotics.

We recently reported the discovery of a 17-kDa Plasmodium yoelii protein expressed in infected hepatocytes and erythrocytes, P. yoelii hepatocyte erythrocyte protein 17 (PyHEP17), and have demonstrated that this protein is a target of protective antibodies and T cells. Here, we report the identification and characterization of the gene encoding this protein and reveal that it is composed of two exons. Immunization of mice with PyHEP17 plasmid DNA induces antibodies, cytotoxic T lymphocytes, and protective immunity directed against the infected hepatocyte. Based on extensive sequence homology, expression pattern, and antigenic cross-reactivity, the Plasmodium falciparum homolog of PyHEP17 is identified as the protein known as exported protein-1 (PfExp-1), also called antigen 5.1, circumsporozoite related antigen, or QF116. Identity between PyHEP17 and PfExp-1 is 37% at the amino acid level (60/161 residues), mapping primarily to two regions within the second exon of 73% (16/22 residues) and 71% (25/35 residues) identity. On this basis, PfExp-1 is proposed as an important component of pre-erythrocytic human malaria vaccines.

The O-linked oligosaccharides attached to human erythrocyte glycophorins were extensively characterized. In addition to the previously described disialylated tetrasaccharide, NeuNAc alpha 2—3Gal beta 1—3 (Neu-NAc alpha 2—6)GalNAcOH and monosialylated trisaccharide, NeuNAc alpha 2—3Gal beta 1—3GalNAcOH, novel trisialylated oligosaccharides were isolated. Methylation analysis, fast atom bombardment-mass spectrometry, and enzymatic degradation were used to elucidate the following novel structures: formula; see text: These results suggest that O-linked oligosaccharides with a disialosyl group, NeuNAc alpha 2—8NeuNAc alpha 2—, may be present in various tissues.