Neurosensory deficit and functional impairment after sagittal ramus osteotomy: a long-term follow-up study.

PURPOSE: This study evaluated persistent neurosensory deficit (NSD) and functional sensory deficit (FSD) after mandibular bilateral sagittal split osteotomies (BSSO) and their association with patient age at time of operation and eight additional variables. PATIENTS AND METHODS: Eighty-five patients more than 2 years post-BSSO were identified and stratified by age: group 1, 10 to 19 years (n=16); group 2, 20 to 29 years (n=24); group 3, 30 to 39 years (n=30); group 4, older than 40 years (n=15). Mean mandibular advancement, incidence of "bad split," excessive intraoperative bleeding, nerve manipulation, removal of third molars, use of rigid fixation, simultaneous mandibular procedures, and associated systemic disease were documented for each group. A questionnaire modified from Zuniga was used to document the presence of persistent (2 years or longer) NSD and FSD. Statistical analysis was performed to determine differences between groups. Logistic regression was used to evaluate each variable and determine its association with persistent NSD and FSD. RESULTS: Persistent NSD by age was: group 1, 81%; group 2, 46%; group 3, 73%; group 4, 87%. The trend of increasing persistence with increasing age was not significant (P=.248). However, persistent FSD with increasing age was highly significant (P=.003). The incidence of FSD in group 4 was statistically greater than in the other groups (P < .001; P < .001; P=.004, respectively). Logistic regression identified patient age and "bad splits" as associated with FSD (P=.003; P=.015, respectively). CONCLUSIONS: The incidence of persistent FSD more than 2 years post-BSSO increases with increasing age in a predictable and highly significant manner. Presurgical counseling should address this issue. FSD is also significantly associated with "bad splits." No other variables were found to be significant.

CASP, a novel, highly conserved alternative-splicing product of the CDP/cut/cux gene, lacks cut-repeat and homeo DNA-binding domains, and interacts with full-length CDP in vitro.

Human CDP/cut and its murine counterpart, cux1/CDP are homeodomain repressor proteins in the family of Drosophila Cut. Northern blot analysis reveals complex alternative splicing, including forms too small to encode the full 1505 amino acid protein. We have characterized a CDP/cut alternatively spliced cDNA (CASP) of 3.4 kb. Human CASP, a predicted 678 amino acid polypeptide, shares 400 amino acids with CDP, but has an alternate N terminal exon of 20 aa, and the C-terminal 258 amino acids diverge from CDP/cut entirely. As the unique C-terminus of CASP lacks the three 'cut-repeats' and homeodomain of CDP/cut, we predict it does not bind DNA. Murine CASP, 96% similar to human, shares these features. Database searches identify homologs in chicken (86% identical to human CASP) and yeast (29% identical to human). Murine CASP mRNA is ubiquitous in mouse tissues and in tissue-culture cell lines. We generated a specific antiserum against the unique C-terminus of CASP, and used this reagent to demonstrate that CASP protein is expressed as an approx. 80 kDa protein in human and murine cells. Co-translation of in vitro-translated CDP and CASP mRNA, followed by immunoprecipitation with specific anti-CASP IgG, shows that CASP polypeptide can from a complex with CDP. Studies of the intron/exon structure of the murine cux/CDP/mCASP locus (>> 100 kb) reveal that the unique 3' exons of CASP are interposed between cut-repeats 2 and 3 of the cux gene. We speculate that a primordial CASP-like gene captured a cut-repeat-homeobox gene to give rise to the eukaryotic Cut/CDP family of proteins.

Repressor activity of CCAAT displacement protein in HL-60 myeloid leukemia cells.

CCAAT displacement protein (CDP)/cut is implicated in several systems as a transcriptional repressor of developmentally regulated genes. In myeloid leukemia cells, CDP/cut binding activity as assayed on the promoter of the phagocyte-specific cytochrome heavy chain gene gp91-phox varies inversely with expression of gp91-phox mRNA. We used two approaches to ascertain whether CDP/cut serves as a repressor of gp91-phox gene expression. First, we used transient transfection assays in 3T3 cells to demonstrate that the CDP/cut binding site from the gp91-phox promoter acts as a negative regulatory element in artificial promoter constructs. Second, we isolated a stable transformant of HL-60 myeloid cells constitutively expressing transfected CDP/cut cDNA. Stable transformants carrying expression vector alone or expressing CDP/cut mRNA were induced to differentiate along the macrophage lineage with phorbol ester or along the neutrophil lineage with dimethyl sulfoxide or retinoic acid/dimethylformamide. Northern blot analysis was used to assess induction of mRNAs encoding gp91-phox, and the myeloid oxidase cytosolic components, p47 and p67. In the stable transformant expressing transfected CDP/cut cDNA, gp91-phox induction was selectively reduced, whereas morphologic differentiation and induction of mRNA for myeloid oxidase components p47 and p67 were unaffected. These data provide persuasive evidence that CDP/cut acts to repress the gp91-phox gene.

Alternative RNA splicing generates transcripts encoding a thorax-specific isoform of Drosophila melanogaster myosin heavy chain.

Genomic and cDNA sequencing studies show that transcripts from the muscle myosin heavy-chain (MHC) gene of Drosophila melanogaster are alternatively spliced, producing RNAs that encode at least two MHC isoforms with different C termini. Transcripts encoding an MHC isoform with 27 unique C-terminal amino acids accumulate during both larval and adult muscle differentiation. Transcripts for the second isoform encode one unique C-terminal amino acid and accumulate almost exclusively in pupal and adult thoracic segments, the location of the indirect flight muscles. The 3' splice acceptor site preceding the thorax-specific exon is unusually purine rich and thus may serve as a thorax-specific splicing signal. We suggest that the alternative C termini of these two MHC isoforms control myofilament assembly and may play a role in generating the distinctive myofilament organizations of flight muscle and other muscle types.

A constitutively transcribed actin gene is associated with the nuclear matrix in a Drosophila cell line.

The relationship between transcriptional activity and gene association with the nuclear matrix has been investigated in Drosophila melanogaster. The nuclear matrix of Schneider cell line 2 of Drosophila was isolated and observed to conform to expected dimensions in phase contrast and scanning electron microscopic preparations. This structure contains proteins that appear similar to the intact nucleus. High salt extracted nuclei digested with DNase I released 98% of the DNA, whereas digestion with Eco RI released a maximum of 80%. These and other nuclease digestions indicate that satellite DNA as well as some unique sequence DNA are bound to the nuclear matrix. A constitutively transcribed actin gene was enriched in the nuclear matrix bound DNA. Two other nontranscribed genes, a muscle-specific actin gene and the myosin heavy chain gene, showed no enrichment in nuclear matrix DNA.

Drosophila muscle myosin heavy chain encoded by a single gene in a cluster of muscle mutations.

Drosophila muscle myosin heavy chain is encoded by a single-copy gene which is transcribed during both larval and adult development. This myosin gene maps to a chromosomal locus distant from any of the actin genes, but is within a cluster of flight muscle mutations.

Isolation and partial characterization of Drosophila myoblasts from primary cultures of embryonic cells.

We describe a method for preparing highly enriched cultures of Drosophila myoblasts from a heterogeneous cell population derived from gastrulating embryos. Enriched cultures are prepared by plating this heterogeneous population of cells in medium from which much of the free calcium is chelated by ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA). Adhesion of myoblasts to tissue culture plastic is better than that of other cell types when plated in this medium. Data concerning cell identity, timing of S phase, and fusion kinetics document the degree of enrichment for myogenic cells and illustrate their synchronous differentiation in vitro.

Actinomycin D-sensitive periods in the differentiation of Drosophila neurons and muscle cells in vitro.

Observations were made of neuroblasts differentiating into neurons, and myoblasts differentiating into myocytes in cultures of embryonic Drosophila cells. Axons greater than 50 mum long appeared in vitro between 7.5 and 16.5 h, and pulsating myocytes appeared between 12.5 and 23.5 h. Actinomycin D treatment prevented neuroblasts and myoblasts from proceeding in differentiation. Neurons became resistant to actinomycin D when they reached the stage of axon initiation, and axon elongation was not actinomycin D-sensitive. Myocytes were sensitive to actinomycin D until they attained the ability to pulsate, and ongoing pulsations were not halted by the drug. Autoradiographs and controls indicated that actinomycin D prevented uridine incorporation by about 90% but prevented leucine and thymidine incorporation by 6% or less in these cells. The result favour the interpretation that transcription is necessary in neuron differentation up to the point of axon initiation and in myocytes up to the point where pulsations can begin. Since ribosomal RNA synthesis is unnecessary for these differentiations, necessary RNA synthesis would be messenger or transfer RNA, or RNA of unknown identity.

Differentiation of Drosophila cells lacking ribosomal DNA, in vitro.

Drosophila melanogaster embryos that lacked ribosomal DNA were obtained from appropriate crosses. Cells were taken from such embryos before overt differentiation took place and were cultured in vitro. These cells differentiated into neurons and myocytes with the same success as did wild-type controls. Therefore, ribosomal RNA synthesis is not necessary for the differentiation of neurons and myocytes in vitro.

Differentiation of neuromuscular junctions in cultures of embryonic Drosophila cells.

Cultures were prepared of embryonic cells from Drosophila melanogaster. Neurons and myocytes differentiated in vitro from their respective stem cells. Electron microscopy showed that neuromuscular junctions formed where axons contacted myocytes. Electrical stimuli were applied to axons and these caused contractions of innervated myocytes. This is the first report of insect or other invertebrate neuromuscular junctions differentiating in vitro. In addition, this is the first system reported in which the neurons, myocytes, and junctions are completely differentiated in vitro from neuroblasts and myoblasts.