Michael Stern

Five SWI genes are required for expression of the HO gene in yeast

High-frequency mating type interconversion in yeast requires the HO gene, which encodes a site-specific endonuclease that initiates the switching process. We have isolated and analyzed switching-defective mutants. These mutants define five complementation and linkage groups, SWI 1 to SWI 5. We have shown by two assays, Northern hybridization and beta-galactosidase activity in strains containing an HO-lacZ fusion, that mutants defective any SWI gene fail to express the HO gene. In addition, all of the swi mutants exhibit other phenotypes, the most notable being the inviability of double mutants defective in SWI 4 and in either SWI 1, SWI 2 or SWI 3. These results indicate that the SWI genes function in some way as positive regulators of HO expression and have additional cellular roles.

Activation of the Yeast HO Gene by Release from Multiple Negative Controls

Transcription of the yeast HO gene requires five genes, SWI 1, 2, 3, 4, 5. We present evidence that some SWI products activate HO by antagonizing negative regulatory activities encoded by the SIN genes. sin- mutants (defining six genes) were identified because they express HO in the absence of particular SWI products. We argue that SWI5 activates HO by antagonizing SIN3 and that SWI4 activates HO by antagonizing SIN6. HO is expressed in sin3- daughter cells, hence we infer that the SIN3 product represses HO in wild-type daughter cells and that SWI5 and SIN3 are responsible for the cell-lineage-dependent expression of HO. HO is transcribed only when all types of repression are absent: in mother cells, where SWI5 antagonizes SIN3; in late G1, when SWI4 antagonizes SIN6; and in a or alpha cells, where a1-alpha 2 repression is absent.

Stromal fibroblasts influence oral squamous‐cell carcinoma cell interactions with tenascin‐C

In this study we identified tenascin‐C (TN‐C) and one of its integrin receptors, αvβ6, in oral squamous‐cell carcinoma (SCC) specimens. Neither TN‐C nor αvβ6 are expressed in normal oral mucosa. We also studied 2 human oral squamous‐cell carcinoma cell lines: the highly invasive HSC‐3 cells, and the poorly invasive SCC‐25 cells. We determined that adhesion of these cells to TN‐C involves both α2 and αv integrins. Migration on TN‐C by oral SCC cells required fibroblast‐conditioned medium and did not occur in its absence. This migration was blocked by anti‐α2 and anti‐αv antibodies and was partially inhibited by antibodies to hepatocyte growth factor, epidermal growth factor and transforming growth factor‐β1. When seeded on TN‐C, the poorly invasive SCC‐25 cells formed αvβ6‐positive focal contacts; the HSC‐3 cells did not. HSC‐3, SCC‐25 and PTF cells secrete TN‐C into the culture medium, as determined by Western blot. However, when HSC‐3 cells were inoculated into the floor of the mouth of nude mice, only murine TN‐C could be identified in the reactive stroma adjacent to the resulting tumor nests, demonstrating that in vivo, HSC‐3 cells do not secrete TN‐C. Our results demonstrate that αvβ6 and tenascin‐C are neo‐expressed in oral squamous‐cell carcinoma, and that the tumor stromal environment is influential in oral SCC behavior. Int. J. Cancer 72:369–376, 1997.

A Model for Fetal Cleft Lip Repair in Lambs

Fetal wounds heal without inflammation and scar formation. This phenomenon may, in the future, be applicable to human cleft lip and palate repair. However, extensive experimental work must first be done to document the benefits of in utero repair. We developed a large animal model for creation and repair of a complete cleft lip and alveolus using fetal lambs. The cleft lip and alveolus deformity was created in eight 75-day-gestation fetuses (term = 145 days) and either repaired in three layers or left unrepaired. There were four sham-operated fetuses, and all animals were alive at harvest. Repaired, unrepaired, and control fetuses were harvested at 7, 14, 21, and 70 days following surgery. The unrepaired fetuses demonstrated a complete cleft lip and alveolus with an oronasal fistula. The maxilla was asymmetrical, with the greater segment deviated toward the cleft and with decreased anterior maxillary width. In contrast, repaired cleft lip and alveolus animals showed no scar, normal thickness of the lip, and a symmetrical maxilla. Histologic analysis of the repaired wounds showed evidence of tissue regeneration without scar formation. The results of this preliminary study indicate that the fetal lamb cleft lip and alveolus model is technically feasible with an excellent survival rate. Healing occurs without scar formation. In the repaired animals, the maxilla was symmetrical. This model will be used to document facial growth following in utero repair of a cleft lip and alveolus.

Long-term viability of the temporalis muscle/fascia flap used for temporomandibular joint reconstruction.

Temporalis muscle/fascia axial flaps (TFs) were used in 115 temporomandibular joints (TMJs) in 81 patients to correct ankylosis (n = 25 joints), traumatic defects (n = 8), congenital anomalies (n = 4), defects resulting from tumor resection (n = 2), degenerative joint disease (n = 52), autoimmune arthritides (n = 21), and lateral capsule flaccidity (n = 3). The follow-up period ranged from 6 months to 5.5 years. Seven patients (8.6% of the group; 10 TMJs) were reevaluated for recurrent symptoms (pain and decreased motion), with a mean of 1.7 years (range = 1 to 3 years) postoperatively. Four of these patients (seven TMJs) had magnetic resonance imaging (MRI) as part of their diagnostic workup and four patients (five TMJs) required a second operative procedure. This study reports the results of the MRI, intraoperative, and histologic evaluations of the TFs in these seven patients. The MRIs showed vascularized tissue between the condyle and roof of the glenoid fossa in all seven joints examined. The signal was consistent with muscle and/or fat as opposed to scar tissue. All flaps examined at the time of surgery (arthroscopy, n = 1; arthrotomy, n = 4) were in place and had the gross appearance of normal muscle. Histologic examination of biopsies of four flaps indicated the presence of viable muscle with normal-appearing nuclei. The results of this study indicate that the TF does survive when it is carefully dissected and inferiorly based to preserve blood supply.

Fetal Cleft Lip Repair in Rabbits: Long-Term Clinical and Cephalometric Results

Abstract We have developed a model for fetal cleft lip (CL) repair in rabbits. To date, the in utero CL procedure has been performed on 174 fetuses in 98 pregnant does. Details of the model, wound healing characteristics, and early growth results have been published previously. In this study, we report long-term clinical and cephalometric findings in 23 fetuses who underwent the fetal CL procedure, were born alive, and survived until completion of growth. The surgically created and repaired CL in fetal rabbits described here resulted in healing without scar formation. The deformity varies from an incomplete to a severe complete cleft, resembling the clinical spectrum of spontaneous clefts in humans. Cephalometric studies indicate that there were no statistically significant differences in premaxillary width, anterior maxillary length, or anterior and posterior maxillary width among control, unrepaired, and repaired animals. Documentation of this phenomenon in higher animals is necessary before the techniq...

Fetal cleft lip repair in rabbits: Histology and role of hyaluronic acid

This study examines the histologic and biochemical features of wound healing in a cleft lip model in the mid-third-trimester fetal rabbit. At days 1, 2, and 4 after the procedure, control, unrepaired, and repaired fetal heads were obtained, sectioned, and stained for histologic examination. The localization of hyaluronic acid in the wound was documented using a cartilage-derived hyaluronic acid-binding protein. In both repaired and unrepaired wounds, the fetal cleft healed without inflammatory cell infiltration or scar formation. Six months after birth, the repaired cleft showed complete regeneration of muscle across the wound and the collagen fibers were of normal density and orientation. Decreased hyaluronic acid deposition was observed in unrepaired clefts as compared with adjacent tissue; no such difference was detected in repaired clefts. Our findings support the hypothesis that a cleft lip repaired in utero heals without the scarring that accompanies postnatal repair. This may explain the lack of maxillary growth restriction after in utero cleft lip repair.

Purification and crystallization of bovine liver phosphorylase

We have purified and crystallized bovine liver phosphorylase a. Starting from 2.5 kg of liver, we obtain 250 mg of phosphorylase a, with a specific activity of 90 units/mg, representing 15% recovery. SDS polyacrylamide gels show three bands, a 95 kDa band with the same mobility as muscle phosphorylase, and two smaller bands of 55 kDa and 40 kDa, which are probably proteolytic fragments. These fragments remain associated and have native conformation and catalytic activity. Crystals which diffract to 2.8 A resolution, were prepared by the hanging drop method using polyethylene glycol PEG 4000 as precipitant. The crystals were prepared in the presence of activators maltotriose and phosphite and crack when placed in solutions containing the inhibitors glucose and caffeine. This suggests phosphorylase is present in an active conformation.