Molecular elements of the regulatory control of keratin filament modulator AHF/trichohyalin in the hair follicle.

Hairs in mammals undergo well-programmed cyclic development, which is strictly controlled by the surrounding hair follicle cells. Among hair follicle epithelia the inner root sheath (IRS) directly envelops the hair shaft to mechanically support its cyclic growth, but the molecular mechanism underlying its sharp regulation remains obscure. Here, we identify regulatory elements for the expression of the IRS-specific protein AHF (Anagenic Hair Follicle antigen), the putative mouse orthologue of trichohyalin (THH), which plays a key role in the assembly of keratin intermediate filaments (IFs) during the hair cycle. AHF becomes abundantly present in the growing anagenic hair follicle and is suddenly diminished as the tissue enters into the subsequent resting stages under the control of ubiquitin-dependent proteolysis. Using primary human keratinocytes, we found that bone morphogenic protein-4 facilitates THH transcription, and intriguingly, a nuclear lamina component plays a key role in the posttranslational stabilization of THH protein. Silencing of the lamin A/C gene leads to rapi THH degradation, whereas exogenously introduced lamin C, but not lamin A, protects THH from proteolytic elimination. These results shed light on the strict molecular mechanisms which control stage- and compartment-specific IF assemblies in support of the cyclic development of the hair shaft.

Hematoma-directed and ultrasound-guided breast-conserving surgery for nonpalpable breast cancer after Mammotome biopsy.

Stereotactic vacuum-assisted (Mammotome) breast biopsy is a powerful diagnostic tool for detecting microcalcifications on mammography, but it is difficult to remove the targeted lesion precisely when subsequent breast-conserving surgery is to be carried out. We achieved satisfactory results by performing hematoma-directed breast-conserving surgery after stereotactic Mammotome biopsy in seven patients. To identify the exact location of the Mammotome biopsy during the breast-conserving surgery, we created an iatrogenic hematoma in the biopsy cavity using patient's blood. This hematoma was detected easily on intraoperative ultrasonography in all patients, and was palpable as a soft mass in five of the seven patients. The microcalcifications were completely removed in all patients, and no cancer cells were found in the margin surfaces after breast-conserving surgery. There were no complications during the injection of the patient's blood into the biopsy cavity or during the hematoma-directed surgery. We describe this new procedure of hematoma-directed breast-conserving surgery following Mammotome biopsy for nonpalpable cancer with microcalcifications.

Identification of Cys385 in the isolated kinase insertion domain of heme-regulated eIF2 alpha kinase (HRI) as the heme axial ligand by site-directed mutagenesis and spectral characterization.

Heme-regulated eIF2alpha kinase (HRI) is an important enzyme that modulates protein synthesis during cellular emergency/stress conditions, such as heme deficiency in red cells. It is essential to identify the heme axial ligand(s) and/or binding sites to establish the heme regulation mechanism of HRI. Previous reports suggest that a His residue in the N-terminal region and a Cys residue in the C-terminal region trans to the His are axial ligands of the heme. Moreover, mutational analyses indicate that a residue located in the kinase insertion (KI) domain between Kinase I and Kinase II domains in the C-terminal region is an axial ligand. In the present study, we isolate the KI domain of mouse HRI and employ site-directed mutagenesis to identify the heme axial ligand. The optical absorption spectrum of the Fe(III) hemin-bound wild-type KI displays a broad Soret band at around 373nm, while that of the Fe(II) heme-bound protein contains a band at 422nm. Spectral titration studies conducted for both the Fe(III) hemin and Fe(II) heme complexes with KI support a 1:1 stoichiometry of heme iron to protein. Resonance Raman spectra of Fe(III) hemin-bound KI suggest that thiol is the axial ligand in a 5-coordinate high-spin heme complex as a major form. Electron spin resonance (ESR) spectra of Fe(III) hemin-bound KI indicate that the axial ligands are OH(-) and Cys. Since Cys385 is the only cysteine in KI, the residue was mutated to Ser, and its spectral characteristics were analyzed. The Soret band position, heme spectral titration behavior and ESR parameters of the Cys385Ser mutant were markedly different from those of wild-type KI. Based on these spectroscopic findings, we conclude that Cys385 is an axial ligand of isolated KI.

Further evidence for recombination between mouse hemoglobin beta b1 and b2 genes based on the nucleotide sequences of intron, UTR, and intergenic spacer regions.

Nucleotide sequences of the intron regions and UTRs (Untranslated regions) of the hemoglobin beta adult genes, b1 and b2, and of the intergenic spacer region were determined for mouse strains representing the d, p, and w1 hemoglobin haplotypes defined by protein electrophoretic analyses. The hypothesis of recombination of the b1 and b2 genes between the d and w1 haplotypes previously reported in the cDNA nucleotide sequences was confirmed by neighbor-joining analyses of the intron regions and UTRs within the b1 and b2 genes, suggesting that all of the structures of hemoglobin beta adult genes support the hypothesis that the p haplotype was established by hybridization between d and w1 haplotype mice. The resultant recombinant of the p haplotype was found to have a d-like b1 gene and a w1-like b2 gene. In addition to the possible recombination, a break point was suggested around 2-3 kb downstream of the b1 gene within the intergenic spacer region, despite the absence of clear properties that could stimulate the recombination machinery. Some large insertions or deletions (indels) specific to the p or d haplotypes were located within the intergenic spacer region, in which the 1010-bp indel specific to the p haplotype was shared by all examined strains representing the p haplotype.

Clinical experience of weekly paclitaxel-based treatment as preoperative chemotherapy for patients with primary breast cancer.

BACKGROUND: Paclitaxel is an effective agent in the treatment of metastatic breast cancer. The aim of this study was to evaluate the safety and efficacy of weekly paclitaxel-based preoperative chemotherapy in patients with large operable breast cancer. METHODS: Patients initially received paclitaxel as a 3-hour infusion at 175 mg/m2. Three weeks after initial administration, two cycles of three weeks of paclitaxel 80 mg/m2 over a 1 hour infusion followed by a one week break were given. Of 22 patients, 9 had stage II (tumor diameter greater than 3 cm), 4 stage III A, 7 stage III B, and 2 stage IV (with ipsilateral supraclavicular lymph node metastasis) cancer, respectively. RESULTS: Excluding stage IV patients, the overall response rate to paclitaxel chemotherapy was 80%. Four of the 20 patients (20%) showed a clinical complete response (cCR). Two of these showed pathologic complete response and the other 2 had only the ductal component remaining. The primary tumor response and axillary lymph node downstaging following preoperative chemotherapy tended to be related in 16 patients with clinically positive nodes. Breast conserving surgery was performed as a result of downstaging in the 9 stage II patients. Grade 3 neutropenia occurred in one patient when 175 mg/m2 of paclitaxel was administered, but no serious side effects developed during the weekly administration of paclitaxel. CONCLUSION: The use of weekly paclitaxel-based preoperative chemotherapy appears to yield a significant anti-tumor effect without inducing serious drug-related adverse effects. Furthermore, the effectiveness of this treatment appears to result in a higher frequency of breast conserving surgery.