Non-invasive transcriptomic analysis using mRNAs in skin surface lipids obtained from children with mild-to-moderate atopic dermatitis.

BACKGROUND: Specimens for analysing the molecular pathology of skin disease are generally obtained through invasive methods, such as biopsy. However, less burdensome methods are desirable for paediatric patients. We recently established a method that comprehensively analyses RNA present in sebum (skin surface lipid-RNAs: SSL-RNAs) using a next-generation sequencer. Using this method, biological information can be obtained from the skin in a completely non-invasive manner. OBJECTIVES: To verify the applicability of the SSL-RNA method for analysis of paediatric skin and analyse the molecular pathology of mild-to-moderate atopic dermatitis (AD) in children. METHODS: We collected sebum specimens from the whole faces of 23 healthy children and 16 children with mild-to-moderate AD (eczema area and severity index (EASI) score: 5.9 ± 2.6) ranging in age from 6 months to 5 years, using an oil-blotting film. We then extracted SSL-RNAs from the samples and performed an AmpliSeq transcriptomic analysis. RESULTS: The expressions of genes related to keratinization (LCE, PSORS1C2, IVL and KRT17), triglyceride synthesis and storage (PLIN2, DGAT2 and CIDEA), wax synthesis (FAR2), ceramide synthesis (GBA2, SMPD3 and SPTLC3), antimicrobial peptides (DEFB1) and intercellular adhesion (CDSN), all of which are related to the skin barrier, are lower in children with AD than in healthy children. The children with AD also have higher expression of CCL17, a Th2-cytokine and an increased Th2-immune response as demonstrated by a gene set variation analysis. Moreover, KRT17 and CCL17 expression levels are significantly correlated with the EASI score. CONCLUSIONS: Molecular changes associated with abnormal immune responses and the epidermal barrier in children with mild-to-moderate AD can be determined using the SSL-RNA method. This non-invasive method could therefore be a useful means for understanding the molecular pathology of paediatric AD.

Angiopoietin-like protein 2 promotes chondrogenic differentiation during bone growth as a cartilage matrix factor.

OBJECTIVE: Chondrocyte differentiation is crucial for long bone growth. Many cartilage extracellular matrix (ECM) proteins reportedly contribute to chondrocyte differentiation, indicating that mechanisms underlying chondrocyte differentiation are likely more complex than previously appreciated. Angiopoietin-like protein 2 (ANGPTL2) is a secreted factor normally abundantly produced in mesenchymal lineage cells such as adipocytes and fibroblasts, but its loss contributes to the pathogenesis of lifestyle- or aging-related diseases. However, the function of ANGPTL2 in chondrocytes, which are also differentiated from mesenchymal stem cells, remains unclear. Here, we investigate whether ANGPTL2 is expressed in or functions in chondrocytes. METHODS: First, we evaluated Angptl2 expression during chondrocyte differentiation using chondrogenic ATDC5 cells and wild-type epiphyseal cartilage of newborn mice. We next assessed ANGPTL2 function in chondrogenic differentiation and associated signaling using Angptl2 knockdown ATDC5 cells and Angptl2 knockout mice. RESULTS: ANGPTL2 is expressed in chondrocytes, particularly those located in resting and proliferative zones, and accumulates in ECM surrounding chondrocytes. Interestingly, long bone growth was retarded in Angptl2 knockout mice from neonatal to adult stages via attenuation of chondrocyte differentiation. Both in vivo and in vitro experiments show that changes in ANGPTL2 expression can also alter p38 mitogen-activated protein kinase (MAPK) activity mediated by integrin α5ß1. CONCLUSION: ANGPTL2 contributes to chondrocyte differentiation and subsequent endochondral ossification through α5ß1 integrin and p38 MAPK signaling during bone growth. Our findings provide insight into molecular mechanisms governing communication between chondrocytes and surrounding ECM components in bone growth activities.

Activation of TLR9 by incorporated pDNA within PEG-coated lipoplex enhances anti-PEG IgM production.

Cationic liposome represents a promising alternative to viral vectors for the delivery of therapeutic genes. For in vivo use, surface modification of the liposome with polyethylene glycol (PEG) is frequently applied to achieve gene-expression in the targeted tissue. However, we have reported that PEG-coated liposomes have induced anti-PEG IgM, which has caused subsequent doses of PEG-coated liposome to be rapidly cleared from blood circulation, and the complexation of pDNA electrostatically associated with liposome surface has enhanced this antibody response. In this study, we investigated how a Toll-like receptor (TLR) might enhance anti-PEG IgM production. PEG-coated pDNA-lipoplex (PDCL) was injected into either wild type, MyD88 (all TLR adaptor protein, independent of TLR3) knock out (KO) or TLR9 KO mice, and the anti-PEG IgM production levels were detected. Attenuated anti-PEG IgM production following the injection of PDCL was observed in both MyD88 and TLR9 KO mice compared to wild type mice, probably due to the abolished induction of cytokines in both MyD88 and TLR9 KO mice. Our results suggest that TLR, exclusively TLR9, signaling plays a potential role in the enhanced anti-PEG IgM production following the injection of PDCL. This result may have important implications for the design and development of an efficient PEG-coated non-viral gene vector.

Endoplasmic reticulum stress-induced apoptosis contributes to articular cartilage degeneration via C/EBP homologous protein.

OBJECTIVE: When endoplasmic reticulum (ER) stress, i.e., the excessive accumulation of unfolded proteins in ER, endangers homeostasis, apoptosis is induced by C/EBP homologous protein (Chop). In osteoarthritis (OA) cartilage, Chop expression and apoptosis increase as degeneration progresses. We investigated the role of Chop in murine chondrocyte apoptosis and in the progression of cartilage degeneration. METHOD: We induced experimental OA in Chop-knockout (Chop(-/-)) mice by medial collateral ligament transection and meniscectomy and compared cartilage degeneration, apoptosis, and ER stress in Chop(-/-)- and wild-type (Chop(+/+)) mice. In our in vitro experiments we treated murine Chop(-/-) chondrocytes with the ER stress inducer tunicamycin (TM) and evaluated apoptosis, ER stress, and chondrocyte function. RESULTS: In vivo, the degree of ER stress was similar in Chop(-/-)- and Chop(+/+) mice. However, in Chop(-/-) mice apoptosis and cartilage degeneration were lower by 26.4% and 42.4% at 4 weeks, by 26.8% and 44.9% at 8 weeks, and by 26.9% and 32.3% at 12 weeks after surgery than Chop(+/+) mice, respectively. In vitro, the degree of ER stress induction by TM was similar in Chop(-/-)- and Chop(+/+) chondrocytes. On the other hand, apoptosis was 55.3% lower and the suppression of collagen type II and aggrecan mRNA was 21.0% and 23.3% less, and the increase of matrix metalloproteinase-13 mRNA was 20.0% less in Chop(-/-)- than Chop(+/+) chondrocytes. CONCLUSION: Our results indicate that Chop plays a direct role in chondrocyte apoptosis and that Chop-mediated apoptosis contributes to the progression of cartilage degeneration in mice.

Targeting the FGF/FGFR axis and its co-alteration allies.

BACKGROUND: We analyzed the FGF/FGFR and co-alteration cancer landscape, hypothesizing that combination therapy might be useful in the presence of co-drivers. MATERIALS AND METHODS: We describe FGF/FGFR-altered pathways, prognosis, and co-alterations [cBioPortal (N = 7574)] and therapeutic outcomes [University of California San Diego Molecular Tumor Board (MTB) (N = 16)]. RESULTS: Patients whose cancers harbored FGF/FGFR alterations (N = 1074) versus those without them (N = 6500) had shorter overall survival (OS) (median: 23.1 versus 26.4 months, P = 0.038) (cBioPortal). Only 6.1% (65/1074 patients) had no pathogenic co-alterations accompanying FGF/FGFR axis abnormalities. The most frequently co-altered pathways/genes involved: TP53 (70%); cell cycle (58%); PI3K (55%); and receptor tyrosine kinases and mitogen-activated protein kinase (MAPK) (65%). Harboring alterations in both FGF/FGFR and in the TP53 pathway or in the cell cycle pathway correlated with shorter OS (versus FGF/FGFR-altered without those co-altered signals) (P = 0.0001 and 0.0065). Four of 16 fibroblast growth factor receptor (FGFR) inhibitor-treated patients presented at MTB attained durable partial responses (PRs) (9, 12, 22+, and 52+ months); an additional two, stable disease (SD) of ≥6 months (13+ and 15 months) [clinical benefit rate (SD ≥ 6 months/PR) = 38%]. Importantly, six patients with cyclin pathway co-alterations received the CDK4/6 inhibitor palbociclib (75 mg p.o. 3 weeks on, 1 week off) and the multikinase FGFR inhibitor lenvatinib (10 mg p.o. daily); three (50%) achieved a PR [9 (ovarian), 12 (biliary), and 52+ months (osteosarcoma)]. Palbociclib and lenvatinib were tolerated well. CONCLUSIONS: FGF/FGFR alterations portend a poor prognosis and are frequently accompanied by pathogenic co-aberrations. Malignancies harboring co-alterations that activate both cyclin and FGFR pathways can be co-targeted by CDK4/6 and FGFR inhibitors.

Effect of chronic kidney disease on excessive daytime sleepiness in Parkinson disease.

BACKGROUND AND PURPOSES: Excessive daytime sleepiness (EDS) is a common sleep disorder in patients with Parkinson disease (PD). Non-ergot dopamine agonists increase the risk of unanticipated sleep episodes. OBJECTIVE: We aimed to assess the influence of renal function on EDS in patients with PD. METHODS: Sixty-two patients treated with ropinirole or pramipexole were recruited for this study. We evaluated the historical and clinical characteristics including the motor symptom rating scales, Epworth Sleepiness Scale (ESS), and estimated glomerular filtration rate (eGFR). An ESS score of 10 or greater was defined as EDS. Participants with eGFR < 60 ml/min/1.73 m(2) were determined to have chronic kidney disease (CKD). Multiple logistic regression analysis was performed to determine the predictive factors of EDS. RESULTS: Chronic kidney disease was found to be a significant predictive factor for EDS in all patients (P = 0.014). We observed a negative correlation between the severity of daytime sleepiness and renal function in patients treated with pramipexole alone (r(s) = -0.637, P < 0.001). CONCLUSIONS: Chronic kidney disease may be a risk factor for EDS, especially in patients treated with pramipexole, which is directly excreted in the urine.

Clonal distribution of enterotoxigenic Staphylococcus aureus on handles of handheld shopping baskets in supermarkets.

AIMS: Shopping carts and handheld shopping baskets in supermarkets are subject to accidental bacterial contamination through contacts with a variety of food. We investigated the prevalence of Staphylococcus aureus on the handles of handheld shopping baskets in four supermarkets distantly located in Osaka district, Japan. METHODS AND RESULTS: Fifty two strains of Staph. aureus were isolated from 760 basket handles. Among these, six strains were positive for staphylococcal enterotoxin B (SEB) production, representing 12% of total. This SEB producer ratio is considerably higher than among Staph. aureus isolated from nasal swabs of the supermarket workers (2%) and from independently collected clinical specimens (4%). These SEB-producing Staph. aureus strains from the basket handles are clonal and belong to ST12. Coagulase typing showed that they are in group VII, which is the most common cause of food poisoning in Japan. Biofilm assays indicated that SEB gene (seb)-positive strains including this clone produced a significantly higher amount of biofilm than seb-negative strains. CONCLUSIONS: The frequent isolation of seb-positive Staph. aureus on shopping basket handles raises the possibility that they could be a hidden reservoir for Staph. aureus with a potential to cause food poisoning and draws attention to the importance of shopping basket sanitation.

A life stage of particle-laden rat dendritic cells in vivo: their terminal division, active phagocytosis, and translocation from the liver to the draining lymph.

Initiation of an adoptive immune response against pathogenic organisms, such as bacteria and fungi, may involve phagocytic activity of dendritic cells (DC) or their immature precursors as a prelude to antigen processing and presentation. After intravenous injection of rats with particulate matter, particle-laden cells were detected in the peripheral hepatic lymph. Since it has been known there is a constant efflux of DC from nonlymphoid organs into the draining peripheral lymph, we examined whether these particle-laden cells belonged to the DC or macrophage lineage. The majority of particle-laden cells in lymph showed immature monocyte-like cytology, and the amount of ingested particles was small relative to typical macrophages. We identified these particle-laden cells as DC based on a number of established criteria: (a) they had a phenotype characteristic of rat DC, that is, major histocompatibility complex class Ihigh+ and IIhigh+, intercellular adhesion molecule 1+ and 80% positive with the rat DC-specific mAb OX62; (b) they showed strong stimulating capacity in primary allogeneic mixed leukocyte reaction; (c) in vitro, they had little phagocytic activity; and (d) the kinetics of translocation was similar to that of lymph DC in that they migrated to the thymus-dependent area of the regional nodes. Furthermore, bromodeoxyuridine feeding studies revealed that most of the particle-laden DC were recently produced by the terminal division of precursor cells, at least 45% of them being <5.5 d old. The particle-laden DC, defined as OX62+ latex-laden cells, were first found in the sinusoidal area of the liver, in the liver perfusate, and in spleen cell suspensions, suggesting that the site of particle capture was mainly in the blood marginating pool. It is concluded that the particle-laden cells in the hepatic lymph are recently produced immature DC that manifest a temporary phagocytic activity for intravascular particles during or after the terminal division and that the phagocytic activity is downregulated at a migratory stage when they translocate from the sinusoidal area to the hepatic lymph.

Fetal liver T cell receptor gamma/delta+ T cells as cytotoxic T lymphocytes specific for maternal alloantigens.

We have established fetal liver-derived T cell receptor (TCR) gamma/delta+, CD3+ T cell lines that are cytotoxic for maternal T cells. Fetal liver-derived lymphoid progenitors yielded predominantly TCR-gamma/delta+ cell clusters when cultured on fetal bone marrow-derived stromal cells in the presence of a cytokine cocktail under magnetic force. These tightly adherent clusters were cloned by limiting dilution and the resulting cell lines analyzed for phenotype and function. Six of eight TCR-gamma/delta lines from 8-9.5-wk gestation fetuses were V delta 2+ as compared with zero of eight lines from later stages of gestation (10 and 15 wk), where all the lines were V delta 1+. In cytotoxicity assays, these TCR-gamma/delta+, CD3+, CD4-, and CD8+ or CD8- long-term cultured lymphoid cells (LLC) were killer cells active against the class I antigens on maternal T cells. Of the cell lines, the CD8+ TCR-gamma/delta+ LLC had the highest levels of killer activity. Thus fetal liver TCR-gamma/delta+ T cells may play a crucial role in protection against invading maternal T cells generated in the feto-maternal interaction.

Clinically significant micafungin resistance in Candida albicans involves modification of a glucan synthase catalytic subunit GSC1 (FKS1) allele followed by loss of heterozygosity.

OBJECTIVES: To determine the mechanism of intermediate- and high-level echinocandin resistance, resulting from heterozygous and homozygous mutations in GSC1 (FKS1), in both laboratory-generated and clinical isolates of Candida albicans. METHODS: The DNA sequences of the entire open reading frames of GSC1, GSL1 (FKS3) and RHO1, which may contribute to the beta-1,3-glucan synthase of a micafungin-susceptible strain and a resistant clinical isolate, were compared. A spontaneous heterozygous mutant isolated by selection for micafungin resistance, and a panel of laboratory-generated homozygous and heterozygous mutants that possessed combinations of the echinocandin-susceptible and -resistant alleles, or mutants with individual GSC1 alleles deleted, were used to compare levels of echinocandin resistance and inhibition of glucan synthase activity. RESULTS: DNA sequence analysis identified a mutation, S645P, in both alleles of GSC1 from the clinical isolate. GSL1 had two homozygous amino acid changes and five non-synonymous nucleotide polymorphisms due to allelic variation. The predicted amino acid sequence of Rho1p was conserved between strains. Reconstruction of the heterozygous (S645/S645F) and homozygous (S645F/S645F) mutation showed that the homozygous mutation conferred a higher level of micafungin resistance (4 mg/L) than the heterozygous mutation (1 mg/L). Exposure of the heterozygous mutant to micafungin resulted in a loss of heterozygosity. Kinetic analysis of beta-1,3-glucan synthase activity showed that the homozygous and heterozygous mutations gave echinocandin susceptibility profiles that correlated with their MIC values. CONCLUSIONS: A homozygous hot-spot mutation in GSC1, caused by mutation in one allele and then loss of heterozygosity, is required for high-level echinocandin resistance in C. albicans. Both alleles of GSC1 contribute equally and independently to beta-1,3-glucan synthase activity.

Expression of Peroxisome Proliferator-activated Receptor-γ in the Kidneys of Cats with Chronic Kidney Disease.

Peroxisome proliferator-activated receptor (PPAR)-γ plays an important role in various cellular functions and its activation exerts protective effects in kidney diseases. In the present study, chronic kidney disease in cats was examined, and changes in renal expression of PPARγ were observed by use of immunohistochemistry. In normal kidneys, nuclei of the superficial cortical tubules, medullary tubules and glomerular cells expressed PPARγ. The vascular walls (tunica media) also showed positive expression. In diseased kidneys, the expression of PPARγ varied between the cases. Some cases showed strong expression, while others had weak expression. PPARγ expression in the nuclei of infiltrating mononuclear cells was also detected in over half of the cases. Although there was no significant relationship between the expression of renal PPARγ and the severity of kidney disease, the fact that there were many cases where the expression of renal PPARγ was reduced was an important finding, and might be one of the possible mechanisms underlying feline chronic kidney diseases.

Increased insulin demand promotes while pioglitazone prevents pancreatic beta cell apoptosis in Wfs1 knockout mice.

AIMS/HYPOTHESIS: The WFS1 gene encodes an endoplasmic reticulum (ER) membrane-embedded protein called Wolfram syndrome 1 protein, homozygous mutations of which cause selective beta cell loss in humans. The function(s) of this protein and the mechanism by which the mutations of this gene cause beta cell death are still not fully understood. We hypothesised that increased insulin demand as a result of obesity/insulin resistance causes ER stress in pancreatic beta cells, thereby promoting beta cell death. METHODS: We studied the effect of breeding Wfs1 ( -/- ) mice on a C57BL/6J background with mild obesity and insulin resistance, by introducing the agouti lethal yellow mutation (A ( y ) /a). We also treated the mice with pioglitazone. RESULTS: Wfs1 ( -/- ) mice bred on a C57BL/6J background rarely develop overt diabetes by 24 weeks of age, showing only mild beta cell loss. However, Wfs1 ( -/- ) A ( y ) /a mice developed selective beta cell loss and severe insulin-deficient diabetes as early as 8 weeks. This beta cell loss was due to apoptosis. In Wfs1 ( +/+ ) A ( y ) /a islets, levels of ER chaperone immunoglobulin-binding protein (BiP)/78 kDa glucose-regulated protein (GRP78) and phosphorylation of eukaryotic translation initiation factor 2, subunit alpha (eIF2alpha) apparently increased. Levels of both were further increased in Wfs1 ( -/- ) A ( y ) /a murine islets. Electron micrography revealed markedly dilated ERs in Wfs1 (-/-) A ( y ) /a murine beta cells. Interestingly, pioglitazone treatment protected beta cells from apoptosis and almost completely prevented diabetes development. CONCLUSIONS/INTERPRETATION: Wfs1-deficient beta cells are susceptible to ER stress. Increased insulin demand prompts apoptosis in such cells in vivo. Pioglitazone, remarkably, suppresses this process and prevents diabetes. As common WFS1 gene variants have recently been shown to confer a risk of type 2 diabetes, our findings may be relevant to the gradual but progressive loss of beta cells in type 2 diabetes.

The oncogenic mutation in the pleckstrin homology domain of AKT1 in endometrial carcinomas.

BACKGROUND: The phosphatidylinositol 3'-kinase (PI3K)-AKT pathway is activated in many human cancers and plays a key role in cell proliferation and survival. A mutation (E17K) in the pleckstrin homology domain of the AKT1 results in constitutive AKT1 activation by means of localisation to the plasma membrane. The AKT1 (E17K) mutation has been reported in some tumour types (breast, colorectal, ovarian and lung cancers), and it is of interest which tumour types other than those possess the E17K mutation. METHODS: We analysed the presence of the AKT1 (E17K) mutation in 89 endometrial cancer tissue specimens and in 12 endometrial cancer cell lines by PCR and direct sequencing. RESULTS: We detected two AKT1 (E17K) mutations in the tissue samples (2 out of 89) and no mutations in the cell lines. These two AKT1 mutant tumours do not possess any mutations in PIK3CA, PTEN and K-Ras. INTERPRETATION: Our results and earlier reports suggest that AKT1 mutations might be mutually exclusive with other PI3K-AKT-activating alterations, although PIK3CA mutations frequently coexist with other alterations (such as HER2, K-Ras and PTEN) in several types of tumours.

Expression of a human hepatocyte growth factor/scatter factor cDNA in MDCK epithelial cells influences cell morphology, motility, and anchorage-independent growth.

The addition of exogenous hepatocyte growth factor (HGF)/scatter factor (SF) to MDCK epithelial cells results in fibroblastic morphology and cell motility. We generated HGF/SF producing MDCK cells by transfection with an expression plasmid containing human HGF/SF cDNA. Production of HGF/SF by these cells induced a change from an epithelial to a fibroblastic morphology and increased cell motility. In addition, the HGF/SF producing cells acquired efficient anchorage-independent growth in soft agar but did not form tumors in nude mice. The morphological change and the stimulation of the anchorage-independent growth were prevented by anti-HGF/SF antibody, suggesting that the factor is secreted and then exerts its effects through cell surface receptors.

Cytoplasmic filaments in developing and adult vertebrate smooth muscle.

An extensive study of adult and developing smooth muscle has revealed the widespread occurrence of a distinct filament with an average diameter of about 100 A (termed the 100 A filament). Unlike that of myofilaments, their appearance in longitudinal section is uniform, but in transverse section they have a round profile, occasionally exhibiting a less electron-opaque core. The 100 A filaments are almost invariably preserved under a variety of fixation procedures, whereas myofilaments, particularly the thicker filaments, are preserved inconsistently. The 100 A filaments appear to be randomly oriented throughout the cytoplasm, either singly or in small groups, although they are sometimes concentrated in the juxtanuclear region of the smooth muscle cells. The intimate association of 100 A filaments with dark bodies, in both developing and adult smooth muscle cells, may indicate that these filaments either play a role in dark body formation or, at least, constitute a part of the dark body. The 100 A filaments are conspicuous in developing smooth muscle cells and occasionally form networks or clusters; they appear to decrease in relative number as maturation proceeds, but considerable numbers are still present in adult tissue.

Induction of in vitro differentiation of mouse embryonal carcinoma (F9) and erythroleukemia (MEL) cells by herbimycin A, an inhibitor of protein phosphorylation.

Herbimycin A is one of the benzenoid ansamycin antibiotics isolated from a culture of Streptomyces species (Omura, S., A. Nakagawa, and N. Sadakane. 1979. Tetrahedron Lett. 1979: 4323-4326). Recent studies have shown that the antibiotic not only inhibits the phosphorylation of p60src in Rous sarcoma virus- (RSV) infected cells, but also reverses the cellular phenotypes acquired by transfection with tyrosine kinase oncogenes (Uehara, Y., M. Hori, T. Takeuchi, and H. Umezawa. 1985. Jpn. J. Cancer Res. 76:672-675; Uehara, Y., M. Hori, T. Takeuchi, and H. Umezawa. 1986. Mol. Cell. Biol. 6: 2198-2206; Uehara, Y., Y. Murakami, S. Mizuno, and S. Kawai. 1988. Virology. 164: 294-298). These studies and other evidence indicate that the antibiotic inhibits a reaction(s) closely associated with the function of cellular tyrosine kinases. We have found that herbimycin A is an effective inducing agent capable of triggering differentiation in two typical mouse in vitro differentiation systems, which have been considered to be quite different in their mechanism of induction: endoderm differentiation of embryonal carcinoma (F9) cells and terminal erythroid differentiation of erythroleukemia (MEL) cells. The results suggest that there is a common step in the intracellular differentiation cascade which is, directly or indirectly, associated with phosphorylation at specific (tyrosine) residues of cellular proteins. The significance of this finding with respect to the molecular mechanism of in vitro differentiation is discussed.

Fine structure of smooth muscle cells grown in tissue culture.

The fine structure of smooth muscle cells of the embryo chicken gizzard cultured in monolayer was studied by phase-contrast optics and electron microscopy. The smooth muscle cells were irregular in shape, but tended to be elongate. The nucleus usually contained prominent nucleoli and was large in relation to the cell body. When fixed with glutaraldehyde, three different types of filaments were noted in the cytoplasm: thick (150-250 A in diameter) and thin (30-80 A in diameter) myofilaments, many of which were arranged in small bundles throughout the cytoplasm and which were usually associated with dark bodies; and filaments with a diameter of 80-110 A which were randomly orientated and are not regarded as myofilaments. Some of the aggregated ribosomes were helically arranged. Mitochondria, Golgi apparatus, and dilated rough endoplasmic reticulum were prominent. In contrast to in vivo muscle cells, micropinocytotic vesicles along the cell membrane were rare and dense areas were usually confined to cell membrane infoldings. These cells are compared to in vivo embryonic smooth muscle and adult muscle after treatment with estrogen. Monolayers of cultured smooth muscle will be of particular value in relating ultrastructural features to functional observations on the same cells.

Differential competition with cytotoxic agents: an approach to selectivity in cancer chemotherapy.

An approach to increasing the selectivity of cancer chemotherapeutic agents is presented in which noncytotoxic competitive substrates are used to discern the differences in structural requirements for transport of cytotoxic agents between tumor cells and a sensitive host tissue, the hematopoietic precursor cells of the bone marrow. Examples are given for two such systems, one responsible for the transport of nucleosides and another for the transport of amino acids. Cytidine is twice as effective in reducing the toxicity of showdomycin for murine bone marrow cells in culture as it is for murine L1210 leukemia cella. Conversely, homoleucine is twice as effective in reducing the toxicity of melphalan for L1210 cells as it is for bone marrow cells. These observations can serve as a basis for the development of bone marrow protective agents and for the design of cytotoxic agents that may be preferentially transported into tumor cells.

Breed differentiation among Japanese native chickens by specific skull features determined by direct measurements and computer vision techniques.

1. Inter-breed morphological comparisons were made among 11 breeds of Japanese native chickens (Gifujidori, Hinaidori, Shokoku, Totenko, Tomaru, Satsumadori, Shamo, Koshamo, Koeyoshi, Chabo and Nagoya), White Leghorn, broiler chickens (Chunky) and red junglefowl collected in the Philippines, based on results of direct measurements and analysis by computer vision techniques of the skull. 2. Analysis of direct measurements identified two groups of chicken: a small type that included the Chabo, Koshamo, red junglefowl, Gifujidori and Shokoku and a large type that included the remaining breeds studied. These groupings were made based on size determined both in the first (PC1) and second principal component (PC2). The greatest length of the cranium and condylobasal length greatly contributed to the morphological differences between these two groups. 3. Analysis by computer vision techniques, however, identified three groups: the Bantam group (which includes red junglefowl), Shokoku group and Shamo group. White Leghorn clustered within the Shokoku group while the broiler chicken belonged to the Shamo group. The region around the junction of the neural cranium and the visceral cranium contributed greatly to the morphological differences among breeds, both in the PC1 and PC2.

Herbimycin A, a pp60c-src tyrosine kinase inhibitor, inhibits osteoclastic bone resorption in vitro and hypercalcemia in vivo.

Since absence of expression of the c-src gene product in mice indicates that the pp60c-src tyrosine kinase is required and essential for osteoclastic bone resorption, we tested the effects of the antibiotic herbimycin A, which is an inhibitor of pp60c-src on osteoclastic bone resorption in vitro and on hypercalcemia in vivo. We examined the effects of herbimycin A on the formation of bone resorbing osteoclasts in mouse long-term marrow cultures, on isolated rodent osteoclasts and on bone resorption in organ cultures of fetal rat long bones stimulated by parathyroid hormone. We found that herbimycin A in concentrations of 1-100 ng/ml inhibited bone resorption in each of these systems. We determined the effects of herbimycin A (100 ng/ml) on src tyrosine kinase activity in mouse marrow cultures and found that it was decreased. Herbimycin A also decreased elevated blood calcium levels that were induced either by repeated subcutaneous injections of recombinant human interleukin-1 alpha or by a human tumor. There was no evidence for toxicity in any of these culture systems or in mice treated with herbimycin A. A different tyrosine kinase inhibitor that does not inhibit pp60c-src was used as a control and caused none of these effects. These data suggest that pp60c-src tyrosine kinase inhibitors may be useful pharmacologic inhibitors of osteoclastic bone resorption and hypercalcemia.