Meta-analysis of sentinel lymph node biopsy in breast cancer using the magnetic technique.

BACKGROUND: The standard for sentinel lymph node biopsy (SLNB), the dual technique (radiolabelled tracer and blue dye), has several drawbacks. A novel magnetic technique without these drawbacks has been evaluated in a number of clinical trials. It uses a magnetic tracer and a handheld magnetometer to identify and excise sentinel lymph nodes. A systematic review and meta-analysis was performed to assess the performance and utility of the magnetic in comparison to the standard technique. METHODS: MEDLINE, PubMed, Embase and the Cochrane online literature databases were used to identify all original articles evaluating the magnetic technique for SLNB published up to April 2016. Studies were included if they were prospectively conducted clinical trials comparing the magnetic with the standard technique for SLNB in patients with breast cancer. RESULTS: Seven studies were included. The magnetic technique was non-inferior to the standard technique (z = 3·87, P < 0·001), at a 2 per cent non-inferiority margin. The mean identification rates for the standard and magnetic techniques were 96·8 (range 94·2-99·0) and 97·1 (94·4-98·0) per cent respectively (risk difference (RD) 0·00, 95 per cent c.i. -0·01 to 0·01; P = 0·690). The total lymph node retrieval was significantly higher with the magnetic compared with the standard technique: 2113 (1·9 per patient) versus 2000 (1·8 per patient) (RD 0·05, 0·03 to 0·06; P = 0·003). False-negative rates were 10·9 (range 6-22) per cent for the standard technique and 8·4 (2-22) per cent for the magnetic technique (RD 0·03, 0·00 to 0·06; P = 0·551). The mean discordance rate was 3·9 (range 1·7-6·9) per cent. CONCLUSION: The magnetic technique for SLNB is non-inferior to the standard technique, with a high identification rate but with a significantly higher lymph node retrieval rate.

Speciation in ninespine stickleback: reproductive isolation and phenotypic divergence among cryptic species of Japanese ninespine stickleback.

Although similar patterns of phenotypic diversification are often observed in phylogenetically independent lineages, differences in the magnitude and direction of phenotypic divergence have been also observed among independent lineages, even when exposed to the same ecological gradients. The stickleback family is a good model with which to explore the ecological and genetic basis of parallel and nonparallel patterns of phenotypic evolution, because there are a variety of populations and species that are locally adapted to divergent environments. Although the patterns of phenotypic divergence as well as the genetic and ecological mechanisms have been well characterized in threespine sticklebacks, Gasterosteus aculeatus, we know little about the patterns of phenotypic diversification in other stickleback lineages. In eastern Hokkaido, Japan, there are three species of ninespine sticklebacks, Pungitius tymensis and the freshwater type and the brackish-water type of the P. pungitius-P. sinensis species complex. They utilize divergent habitats along coast-stream gradients of rivers. Here, we investigated genetic, ecological and phenotypic divergence among three species of Japanese ninespine sticklebacks. Divergence in trophic morphology and salinity tolerance occurred in the direction predicted by the patterns observed in threespine sticklebacks. However, the patterns of divergence in armour plate were different from those previously found in threespine sticklebacks. Furthermore, the genetic basis of plate variation may differ from that in threespine sticklebacks. Because threespine sticklebacks are well-established model for evolutionary research, the sympatric trio of ninespine sticklebacks will be an invaluable resource for ecological and genetic studies on both common and lineage-specific patterns of phenotypic diversification.

The reeler gene encodes a protein with an EGF-like motif expressed by pioneer neurons.

We have identified a strong candidate cDNA for the mouse reeler gene. This 5 kb transcript encodes a 99.4 kD protein consisting of 881 amino acids and possessing two EGF-like motifs. We assayed two independent mutant alleles--'Jackson reeler', which has a deletion of the entire gene, and 'Orleans reeler' which exhibits a 220 bp deletion in the open reading frame, including the second EGF-like motif and resulting in a frame shift. In situ hybridization reveals that the transcript is detected exclusively in the pioneer neurons which guide neuronal cell migration along the radial array. Our findings offer an explanation for how the reeler mutant phenotype causes a disturbance of the complex architecture of the neuronal network.

A novel methodology for analysis of cell distribution in chimeric mouse organs using a strain specific antibody.

Chimeric animals are very useful for analysis of cell lineage, homeostasis in tissue architecture, and cell-cell interactions during both organogenesis and carcinogenesis. However, there is not a generally effective means for marking cells of chimeric mice. We have therefore developed a polyclonal antibody that is useful for this purpose. This antibody specifically recognizes those cells derived from C3H strain mice. The specificity of this antibody was checked by both immunoblotting and immunoadsorption methods. The antigens were immunohistochemically detected in cytoplasm of both epithelial and mesenchymal cells of C3H/HeN strain mouse in many different organs, but not the corresponding cell types from BALB/c or C57BL/10 or several other mouse strains. The validity of these antibodies as markers for C3H cells was further checked by tissue recombination experiments and in mixed cultures of mouse and rat cells. In each case the antibody recognized only the C3H mouse cells. Next, chimeric mice were prepared between strains C3H/HeN and BALB/c, and C3H/HeN and C57BL/10 mice. Chimeras 2-mo old were examined for antigen distribution using the indirect immunofluorescence method. Many tissues in chimeric mice were composed of cells that were both stained and unstained by the anti-C3H specific antigen. The chimeric patterns were classified into four types, A-D. In well-defined structural units such as intestinal crypts, small intestinal villi, kidney convoluted tubules, exocrine gland acini, ovarian follicles, thyroid gland follicles, stomach glands, adrenal cortex, lingual papillae, etc., (A) each unit was composed entirely of either positive or negative cells, or else (B) in some organs each unit was composed of both types of cells. In the uniform tissues without such distinguishable units, such as stratified squamous epithelium, mesenchymal tissue, corpora lutea, pituitary gland, Islets of Langerhans, adrenal medulla etc., (C) the tissue was composed of definite small cell groups made entirely of either positive or negative cells, or else (D) the tissue was composed of both types of cells which were intermingled with one another. These findings strongly suggest that the chimeric patterns demonstrated here reflect the cell proliferative unit in each tissue. This cell marker system has proven useful for analysis of cell lineage and cell renewal systems in many organs of chimeric mice.

Explosive deep water basalt in the sumisu backarc rift.

Eruption of 1-million-year-old tholeiitic basalt >1800 meters below sea level (>18 megapascals) in a backarc rift behind the Bonin arc produced a scoriaceous breccia similar in some respects to that formed during subaerial eruptions. Explosion of the magma is thought to have produced frothy agglutinate which welded either on the sea floor or in a submarine eruption column. The resulting 135-meter-thick pyroclastic deposit has paleomagnetic inclinations that are random at a scale of <2.5 meters. High magmatic water content, which is about 1.3 percent by weight after vesiculation, contributed to the explosivity.

[Lung cancer operation in situs inversus totalis patient].

Situs inversus totalis represents a complete mirror image anatomy of the normal arrangement of the thoracic and abdominal viscera. This rare condition may pose possible surgical problems due to anatomical abnormality. There were few reports of surgical treatment for lung cancer patient with situs inversus totalis. In this case report, we describe a 74-year-old patient with situs inversus totalis and primary lung cancer who underwent successful left upper lobectomy and systemic lymph node dissection. For this rare condition, detail preoperative evaluation of mirror image anatomy with computed tomography and bronchofiber optic examination was thought to be a key to carry out safe operative procedure.

A hyperthermostable bacterial histone-like protein as an efficient mediator for transfection of eukaryotic cells.

Gene delivery has shown potential in a variety of applications, including basic research, therapies for inborn genetic defects, cancer, AIDS, tissue engineering, and vaccination. Most available systems have serious drawbacks, such as safety hazards, inefficiency under in vivo-like conditions, and expensive production. When using naked DNA, for instance, a large amount of ultrapure DNA has to be applied as a result of degradation by nucleases. Similarly, the use of eukaryotic histones, synthetic peptides, or peptide nucleic acids may be limited by high production costs. We have demonstrated a biotechnologically feasible and economical approach for gene delivery using the histone-like protein from the hyperthermostable eubacterium Thermotoga maritima, TmHU as an efficient gene transfer reagent. HU can be easily isolated from recombinant Escherichia coli, is extraordinarily stable, and protects dsDNA from thermal denaturation. This study demonstrates its use as an inexpensive tool for gene delivery.

Changes in mRNAs encoding steroidogenic acute regulatory protein, steroidogenic enzymes and receptors for gonadotropins during spermatogenesis in rainbow trout testes.

In vertebrates, sperm development and maturation are directly regulated by gonadal steroid hormone secretion. The relationships among the expression of genes encoding steroidogenic proteins and receptors for gonadotropins, and testicular steroid production have not yet been comprehensively determined in male teleosts. In this study, the changes in levels of mRNAs encoding follicle-stimulating hormone (FSH) receptor, luteinizing hormone (LH) receptor, steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage, 3beta-hydroxysteroid dehydrogenase/delta5-4-isomerase, cytochrome P450 17alpha-hydroxylase/17,20-lyase, cytochrome P450 11beta-hydroxylase, 11beta-hydroxysteroid dehydrogenase and 20beta-hydroxysteroid dehydrogenase were determined by real-time, quantitative PCR assays and related to changes in serum steroid levels throughout the reproductive cycle in male rainbow trout. Serum 11-ketotestosterone and 17alpha,20beta-dihydroxy-4-pregnen-3-one levels were measured by RIA. Although the pattern of change in the mRNA levels for the enzymes was variable, the increases in steroidogenic enzyme mRNAs started prior to a significant increase of serum steroid levels. The patterns of transcript levels of FSH and LH receptors suggest that changes in StAR and steroidogenic enzyme transcripts are largely mediated by the FSH receptor during early and mid-spermatogenesis and by the LH receptor during late spermatogenesis and spermiation. Levels of StAR (10-fold) and P450 17alpha-hydroxylase/17,20-lyase (sevenfold) transcripts changed with the greatest magnitude and were closely related to the changes in serum steroids, suggesting that changes in StAR and P450 17alpha-hydroxylase/17,20-lyase abundance are likely to be the major influences on overall steroidogenic output during the reproductive cycle in male rainbow trout.

Strain specific sensitivity to diethylnitrosamine-induced carcinogenesis is maintained in hepatocytes of C3H/HeN in equilibrium with C57BL/6N chimeric mice.

The C3H/HeN (C3H) and C57BL/6N (C57) mouse strains are known, respectively, for their high and low susceptibility to both spontaneous and chemically induced hepatocarcinogenesis. The present study was aimed at elucidating whether this difference is dependent on intrinsic features of the target hepatocytes or in the in vivo milieu and associated growth promoting factors to which the cells are exposed. C3H in equilibrium with C57 chimeric mice were produced and given injections of diethylnitrosamine (20 microgram/g body weight) at the age of 15 days. The animals were sacrificed 6 or 9 months thereafter, and the numbers and sizes of altered cell lesions were scored. The clonal growth of both cell types was immunohistochemically confirmed using anti C3H-specific antigen antibodies. Quantitative assessment revealed C3H lesions in the chimera livers to be far larger (5:1) than those of C57 derivation and associated with more frequent malignant progression as was evident histologically. Furthermore, foamy change and hyalin body formation, which have been described as characteristics of C3H and C57BL hepatic tumors, respectively, were also featured as differentiative characteristics in lesions of both cell types in chimera mice. Thus, the results clearly demonstrated that the principal mechanism(s) underlying strain difference in diethylnitrosamine-initiated hepatocarcinogenesis exists in the target cells and is not milieu-dependent.

Tissue-specific carcinogenesis in transgenic mice expressing the RET proto-oncogene with a multiple endocrine neoplasia type 2A mutation.

Germ line mutations of the RET proto-oncogene are responsible for the development of multiple endocrine neoplasia type 2A (MEN 2A), an inherited cancer syndrome characterized by medullary thyroid carcinoma, pheochromocytoma, and parathyroid hyperplasia. To study the mechanism of tissue-specific tumor development by RET with a MEN2A (cysteine 634-->arginine) mutation, we generated transgenic mice by introducing the RET-MEN2A gene fused to Moloney murine leukemia virus long terminal repeat. Expression of the transgene and its product was detected at variable levels in a variety of tissues including thyroid, heart, liver, colon, parotid gland, and brain. All of 29 mice analyzed developed thyroid C-cell hyperplasia or medullary carcinoma, accompanying high levels of serum calcitonin. In addition, development of mammary or parotid gland adenocarcinoma was observed in one-half of the transgenic mice. RET dimerization and its complex formation with Shc and Grb2 adaptor proteins were detected in tumor tissues. Unexpectedly, no tumor formation was found in other tissues despite RET-MEN2A expression where RET dimerization was undetectable. Because these tissues but not tumors expressed glial cell line-derived neurotrophic factor family receptor alpha (GFR alpha) at high levels, this suggested that GFR alpha expression may interfere in the dimerization of the RET-MEN2A mutant proteins, leading to tissue-specific tumor development in vivo.

Cerebellar histogenesis as seen in identified cells of normal-reeler mouse chimeras.

The potential contribution of cell-cell interactions and extracellular factors to cytoarchitectonic abnormalities in the cerebellum of the reeler mutant mouse was investigated by forming chimeras between the reeler and normal animals. The strain origin of Purkinje cells, granule cells and Golgi epithelial cells was immunohistologically identified with a strain-specific antibody. We analyzed 16 overt coat color chimeras, 10 reeler <--> C3H and 6 reeler <--> Balb/c. Abnormal behavioral traits of reeler were rescued in all chimeras. However, cerebellar histology was more affected in reeler <--> C3H chimeras than in reeler <--> Balb/c. Purkinje cells from the normal genotype occupy ectopic positions, and reeler genotype cells are arranged appropriately in the same chimeric cerebellum. We also obtained histologically normal chimeras with a significantly high contribution of the reeler genotype in Purkinje cells, Golgi epithelial cells and granule cells. These results clearly indicate that the abnormal cell positioning and cytoarchitecture of neurons and glia in the reeler is caused by a deficiency of extracellular environments, but is not determined cell-autonomously. The present data on chimeric mice suggest that Reelin is one of the important extracellular environmental factors that affects indirectly radial glial cells during cerebellar histogenesis.

In situ hybridization with non-radioactive digoxigenin-11-UTP-labeled cRNA probes: localization of developmentally regulated mouse tenascin mRNAs.

An improved method for in situ hybridization was developed in order to identify the tissue-specific expression of messenger RNA (mRNA) for the novel extracellular matrix glycoprotein, tenascin, during mouse development. Non-radioactive RNA probes were generated by incorporating digoxigenin-11-UTP instead of conventional isotopic labels. Hybridization of anti-sense probes to complementary mRNAs was detected by a chromogenic staining reaction catalyzed by an anti-digoxigenin antibody-alkaline phosphatase conjugate. Markedly improved enhancement of staining was achieved by expanding the complexity of probes and strictly controlling the degree of proteolytic digestion of paraformaldehyde-fixed tissue sections. Six different complementary RNA (cRNA) probes representing most of the tenascin mRNA sequence were prepared. Very weak signals were obtained after single applications of each probe, but strong specific signals were present when all six probes were mixed together. In either case, no signal was found without prior proteolytic digestion of tissue sections with proteinase K. Treatment with increasing concentrations of proteinase K initially resulted in increased sensitivity of signal detection, but extensive digestion resulted in histological sections of poor quality for light microscopy. Optimal conditions varied according to the tissue type examined. In lung, in situ hybridization detected tenascin mRNA in the relatively large cells lining alveolar walls adjacent to type I pneumocytes. In cerebellum, glial cells of the Purkinje cell layer contained tenascin mRNA, but Purkinje cells did not. In both cases, hybridization signals were confined to the cytoplasm of cells, and no extracellular staining was observed. This method provides a promising new tool for analysis of spatio-temporal regulation of tenascin gene expression during embryogenesis and oncogenesis.

Cell mixing during the early development of mouse aggregation chimera.

Two different inbred strain combinations of mouse aggregation chimeras - C3H/HeN (H-2k) x C57BL/6N (H-2b) and C3H/HeN x BALB/cA (H-2d) were used for cell mixing analysis at two points in time - 24 h after aggregation (just prior to transplantation into foster mothers) and 7.5 days post coitum (p.c.). The cell proportion of two H-2 haplotypes at the blastocyst stage was studied using a fluorescence-labeled monoclonal antibody recognizing a C3H-specific alloantigen - CSA (C3H strain-specific antigen) and laser scanning confocal microscopy. The 7.5-day-old chimeras were sectioned and subsequently processed by sensitive biotinylated antibody - avidin peroxidase immunohistochemical technique. Our results showed that 24 h after aggregation (blastocyst stage), there was equal cell mixing and no mouse strain used in the present study was dominant at this time. In 7.5-day-old C3H/HeN x BALB/cA chimeras, cells of both genotypes were intermingled, but the C3H/HeN strain was dominant in all cases. In contrast, the combination C3H/HeN x C57BL/6N clearly showed reduced numbers of C3H/HeN cells (CSA-positive) in 83% of the chimeras evaluated. Generally, CSA positive cells were found only in randomly distributed small distinct areas representing less than 20% of embryonal cells. Surprisingly, the extraembryonal ectoplacental cone was uniformly CSA positive in some C3H/HeN x C57BL/6N chimeras. Furthermore, in 36% of normally implanted chimeras of both strain combinations progressive degeneration was observed. We suggest that the cell mixing pattern as well as the absolute number of cells derived from each strain in the aggregation chimera can be affected by specific immune interactions involving H-2 haplotype combinations of the allogeneic fetus and the fully immunocompetent host organism, at later points in development.

Expression of paternal and maternal mitochondrial HSP70 family, hsc74, in preimplantation mouse embryos.

We have investigated the regulation of gene expression of a novel mitochondrial HSP70 family, hsc74 in preimplantation mouse embryos. We used a monoclonal antibody, anti-CSA, which reacts with only one of strain variants of the hsc74. By immunostaining with anti-CSA antibody, the hsc74 protein was constitutively detected in C3H embryos from 1-cell to blastocyst stage, but no signals were detectable in C57BL/6 embryos. To know the timing of paternal genome expression, we examined the expression of hsc74 in (C57BL/6 x C3H)F1 embryos. No positive signals were detectable in embryos before 8-cell stage. In early 8-cell stage weakly positive signals appeared in the peripheral region of the blastomeres. From late 8-cell stage, the protein was intensively detectable and was persistently expressed in all types of cells. We have also applied a sensitive methodology to distinguish genetic variants of hsc74 from C3H and C57BL/6 by reverse transcription polymerase chain reaction followed by single strand conformation polymorphism analysis. In (C57BL/6 x C3H)F1 embryos, the paternal transcripts were first detected in 4-cell embryos, while the maternal transcripts were constantly detectable. These results indicate that the transcripts and proteins of hsc74 were derived only from the maternal gene from 1-cell to 4-cell stages, and that from 4-cell stage the paternal gene is also transcribed, and the significant increase of the paternally derived protein occurred around late 8-cell stage.

Effect of tenascin-C deficiency on chemically induced dermatitis in the mouse.

Tenascin-C is a large extracellular matrix glycoprotein characterized by its spatiotemporal expression during embryogenesis, carcinogenesis, and wound healing. Many in vitro studies on tenascin-C have revealed its multifunctional properties; however, disruption of the tenascin-C gene did not reveal any obvious abnormalities during development, and its function in vivo remains unclear. Here, we investigated whether tenascin-C is involved in inflammatory dermatitis in adults by studying chemically induced dermatitis in tenascin-C knockout mice. An epicutaneous application of a hapten, 2,4-dinitrofluorobenzene, to the ear skin of BALB/CA mice resulted in inflammation and induced the expression of tenascin-C. In congenic tenascin-C knockout mice, the dermatitis occurred more severely than in wild-type mice; infiltration of polymorphonuclear cells in knockout mice persisted longer than in wild-type mice, and the elastosis-like disorganized extracellular matrix was also seen in the ear. These results suggest that tenascin-C plays a role in vivo in inflammatory responses in the skin, and that the genetic background has profound effects on the function of tenascin-C in mouse dermatitis.

Human peptidylarginine deiminase type III: molecular cloning and nucleotide sequence of the cDNA, properties of the recombinant enzyme, and immunohistochemical localization in human skin.

Peptidylarginine deiminase catalyzes the post-translational modification of proteins through the conversion of arginine to citrulline in the presence of calcium ions. In rodents, peptidylarginine deiminase has been classified into four isoforms, types I, II, III, and IV, which are distinct in their molecular weights, substrate specificities, and tissue localization. Of these isoforms, only type III was detected in epidermis and hair follicles. Although the role of this enzyme in these tissues is not yet clear, indirect data have shown that several structural proteins such as filaggrin, trichohyalin, and keratin are substrates for peptidylarginine deiminase. In this study, we cloned the full-length cDNA of human peptidylarginine deiminase type III (3142 bp) from cultured human keratinocytes by reverse transcription-polymerase chain reaction and by rapid amplification of cDNA ends methods. This cDNA contained a 1995 bp open reading frame encoding 664 amino acids (Mr = 74 770). To explore the physicochemical and enzymatic properties of human peptidylarginine deiminase type III, we constructed a plasmid for producing a recombinant human peptidylarginine deiminase type III in bacteria. The enzymatic characteristics of the recombinant enzyme were very similar to those of the rodent peptidylarginine deiminase type III. The recombinant enzyme showed the catalytic activities toward structural proteins of epidermis and hair follicle, filaggrin and trichohyalin, in which the deiminations maxima of about 60% and 13% arginine residues were observed in filaggrin and trichohyalin, respectively. An immunohistochemical study of human scalp skin with a monospecific anti-peptidyl-arginine deiminase type III antibody revealed that the type III enzyme was localized to the inner root sheath and outer root sheath of hair follicles. Peptidylarginine deiminase type III in the inner root sheath was notable between supramatrix and keratogenous zone and was scarcely detected in cornified hair zone. The enzyme was also expressed in the cuticle layer of hair. On the other hand, expression of the enzyme in the epidermis was very low. These data imply that human peptidylarginine deiminase type III is the predominant isoform in hair follicles and may function as a modulator of hair structural proteins, including trichohyalin during hair and hair follicle formation.

Spatiotemporal changes of fibronectin, tenascin-C, fibulin-1, and fibulin-2 in the skin during the development of chronic contact dermatitis.

In order to elucidate how chronic inflammation affects the organization of the extracellular matrix in the skin, a prolonged allergic contact dermatitis was induced in a mouse by repeated application to the ear of 2,4-dinitrofluorobenzene every 3 d for 66 d. Subsequently, the spatiotemporal changes of fibronectin, tenascin-C, fibulin-1, and fibulin-2 in the skin were examined. In the acute phase of inflammation (day 3-day 12), the amount of fibronectin and tenascin-C increased markedly and were degraded, whereas the amount of fibulin-2 changed slightly. Abundant deposition of tenascin-C was observed in the connective tissue. Fibulin-1 and fibulin-2 distributed as fine fibrils. In contrast, the amounts of fibronectin and tenascin-C decreased and their degradation was suppressed in the chronic phase (day 15-day 66), but the amount of fibulin-2 increased. Tenascin-C was observed mainly at and underneath the epidermal basement membrane. In the subepidermal region, many fibulin-2-positive microfibrils were distributed. The amount and distribution of fibulin-1 did not change markedly in either phase. MMP-like enzymes of 62 kDa, probably activated MMP-2, were upregulated in the chronic phase, whereas components of 92, 85, or 67 kDa were highly induced in the acute phase. These results suggest that chronic inflammation in allergic contact dermatitis is associated with temporal changes in the expression, deposition, and degradation of inducible extracellular matrix components.

Survival of steroid 21-hydroxylase-deficient mice without endogenous corticosteroids after neonatal treatment and genetic rescue by transgenesis as a model system for treatment of congenital adrenal hyperplasia in humans.

The genome of mice with the H-2aw18 haplotype has a deletion of approximately 80 kilobases in the H-2 class III region of chromosome 17. Mice that are homozygous for the mutation die soon after birth. A functional form of steroid 21-hydroxylase (21-OHase) is encoded by the deleted DNA fragment, and H-2aw18 homozygotes are deficient in this enzyme. 21-OHase catalyzes the conversion of progesterone to deoxycorticosterone during adrenal steroidogenesis in mice; therefore, H-2aw18 homozygous mice are unable to synthesize corticosteroids. The deleted region also includes the gene for complement component C4, which has a role in the classical pathway of the complement activation cascade. To clarify the cause of the lethality of the mutation, we first administered either an adrenal homogenate or synthetic steroids to newborn mice; as a result, several H-2aw18 homozygotes were rescued. The results demonstrated that the mutant mice die as the result of a defect in adrenal steroidogenesis. The low efficiency of the rescue by treatment of newborns (16.0% by the adrenal homogenate and 14.8% by the synthetic steroids) suggested that mutant mice should be treated prenatally. Moreover, because the 21-OHase gene is expressed before birth, introduction of a gene for 21-OHase should improve the efficiency of rescue. The results of the murine mutation are similar to those of the inherited human disease known as congenital adrenal hyperplasia, which is caused by steroid 21-hydroxylase deficiency. As a model system for treatment of the human disease by genetic therapy, we used transgenic approaches to introduce a recombinant DNA fragment containing the murine genomic gene for 21-OHase into the mutant mice. We produced four lines of transgenic mice, and in all four transgenic lines, the transgene rescued the lethal mutation. The apparent efficiencies of rescue were 80.2%, 80.0%, 68.7%, and 16.7% for the respective lines of transgenic mice. During the course of our experiments, we also found an unexpected property associated with the role of corticosteroids. The H-2aw18 homozygous mice rescued by neonatal treatment survived for a long period without further treatment. This observation indicates that corticosteroids down-stream of 21-OHase in the pathway for adrenal steroidogenesis are not essential for the survival of mice, except during the period immediately after birth.

Murine tenascin: cDNA cloning, structure and temporal expression of isoforms.

Mouse tenascin (TN)-encoding cDNA clones were isolated from a cDNA library of the 2H6GR mammary tumor cell line. Nucleotide (nt) and deduced amino acid (aa) sequences revealed the characteristic primary structure, which begins with a signal peptide and TN unique sequences, follows with 14 1/2 epidermal growth factor (EGF)-like repeats and 13 fibronectin type-III repeats (FN repeat), and concludes with fibrinogen-homologous sequences. Similar to chicken and human TN, the mouse TN cDNA contains five consecutive insertional FN repeats, as well as eight constitutive FN repeats. Three different cDNA clones that may have been generated by alternative splicing of these insertional FN repeats were identified and characterized. Based upon the deduced as sequence, a polyclonal antibody was produced against a synthetic TN peptide. It specifically recognized two TN isoforms of 230 kDNA and 190 kDa in protein extracts of mouse tissues. The tissue distributions of mouse TN mRNAs, revealed by Northern blot analysis, suggest that there is tissue-specific expression of TN isoforms. Two distinct mRNA transcripts (7 kb and 5.5 kg) were detected in brain, skeletal muscle, digestive tract and bladder, but only one was observed in lung, kidney (7 kg) and thymus (5.5 kg). TN mRNA expression was down-regulated 1 month after birth in most tissues. However, the 5.5-kb transcript persisted in cerebellum, thymus, and colon. The spatial and temporal patterns of TN expression seem to be controlled at the level of transcription, because analysis of various tissues by Western blots showed the same pattern as that seen in Northern blots.

Susceptibility of tenascin to degradation by matrix metalloproteinases and serine proteinases.

The degradation of tenascin purified from human melanoma cells was examined by treatment with matrix metalloproteinases (MMPs) and serine proteinases. Among eight different types of proteinases examined, MMP-1, -3, and -7, cathepsin G and leukocyte elastase could digest tenascin, but MMP-2, MMP-9 and thrombin did not. This suggests that tenascin may be readily catabolized by extracellular matrix-degrading proteinases found in the pathophysiological conditions.