Synthesis, conformation and antiproliferative activity of isothiazoloisoxazole 1,1-dioxides.

Sixteen new isothiazoloisoxazole 1,1-dioxides, one new isothiazolotriazole and one new isothiazolopyrazole have been synthesised by using 1,3-dipolar cycloadditions to isothiazole 1,1-dioxides. One sub-set of these isothiazoloisoxazoles showed low µM activity against a human breast carcinoma cell line, whilst a second sub-set plus the isothiazolotriazole demonstrated an interesting restricted rotation of sterically hindered bridgehead substituents. A thiazete 1,1-dioxide produced from one of the isothiazole 1,1-dioxides underwent conversion into an unknown 1,2,3-oxathiazolin-2-oxide upon treatment with Lewis acids, but was inert towards 1,3-dipoles and cyclopropenones. Six supporting crystal structures are included.

Met signaling regulates growth, repopulating potential and basal cell-fate commitment of mammary luminal progenitors: implications for basal-like breast cancer.

Basal-like breast cancer is an aggressive subtype of mammary carcinoma. Despite expressing basal markers, typical of mammary stem cells, this tumor has been proposed to originate from luminal progenitors, which are downstream of stem cells along the mammary epithelial hierarchy. This suggests that committed luminal progenitors may reacquire basal, stem-like characteristics, but the mechanisms that regulate this transition remain unclear. Using mouse models, we found that luminal progenitors express high levels of the Met receptor for hepatocyte growth factor (HGF), as compared with the other mammary epithelial sub-populations. Constitutive activation of Met led luminal progenitors to attain stem cell properties, including enhanced clonogenic activity in vitro and de novo ability to reconstitute mammary glands in repopulation assays in vivo. Moreover, in response to Met signaling, luminal progenitors gave rise to hyperplastic ductal morphogenesis and preferentially underwent basal lineage commitment at the expense of luminal cell-fate specification. Opposite and symmetric results were produced by systemic pharmacological inhibition of Met. Hence, Met signaling targets luminal progenitors for expansion, impairs their differentiation toward the mature luminal phenotype and enables their commitment toward the basal lineage. These results emphasize a critical role for Met in promoting deregulated proliferation and basal plasticity of normal luminal progenitors in the mammary gland, a complex of events that may be required for sustaining the functional and phenotypic properties of basal-like breast tumors.

Prevalence and predictors of carer burden and depression in carers of patients referred to an old age psychiatric service.

BACKGROUND: Too little is currently known about the prevalence of and risk factors for depression and carer strain among informal carers of community-dwelling elderly mentally ill. This study seeks to assess the prevalence of depression, using the Geriatric Depression Scale-15 (GDS-15), the degree of carer burden/strain, and their risk factors among the primary informal carers of patients referred to our community-based old age psychiatry service. METHODS: A cross-sectional study design was used, with the subjects comprising 100 primary informal carers of patients who live at home and were referred to our service. The main carer measures were the GDS-15 and an adapted version of Gilleard's Strain Scale. Patients were assessed the Clifton Assessment Procedure for the Elderly-Survey version, the GDS-15 and the Mini-mental State Examination. RESULTS: Depression was found in 21% of the carers (a score of 5 or more on the GDS-15). The more problem behaviors identified and the greater the functional impairment of the patient, the higher the strain score deciles and the more likely the carer was to be depressed. Spouses were associated with lower carer strain scores. Patient diagnoses did not affect carer depression or carer strain. CONCLUSION: We found high levels of depression in the primary carers of community-dwelling patients attending an old age psychiatric service. The patients' behavior and their cognitive and functional ability conferred greater risk of carer depression or strain than their diagnosis. These risk factors may help identify carers at risk of strain and depression.

Mammary stem cells and breast cancer.

The mammary gland undergoes major developmental changes during puberty and pregnancy. It is thought that stem cells drive mammary gland development during puberty and are responsible for tissue maintenance as well as the major growth and remodelling that occurs with every pregnancy. The use of sophisticated cell separation procedures has facilitated the prospective isolation of mammary epithelial stem and differentiated cell subpopulations from the mouse mammary gland, while studies of primary human breast cancers have described sub-populations of tumourigenic cells capable of initiating tumour growth in immuno-compromised mice. These potential tumour 'stem cells' constitute an important therapeutic target population with respect to cancer therapy, as these are likely to be the cells which maintain tumour growth. Understanding the origin of these cells, their relationship to breast cancer subtypes, and how and why they differ from normal breast stem cells will lead to a revolution in tumour understanding, treatment and prevention. (Part of a Multi-author Review).

Age-related changes to the surface ultrastructure of the rabbit temporomandibular disc.

The ultrastructural characteristics of the temporomandibular disc were investigated by transmission electron microscopy in rabbits, aged from 7 d to 6 y. The disc is interposed between the head of the mandibular condyle below, and the squamous temporal bone above, within the temporomandibular joint. A single layer of flattened mesenchymal cells covered the articular surface of the disc during early postnatal development. Gradual disappearance of this cell layer was succeeded by the development of an articular surface lamina. The first appearance of the lamina coincided with the transition from suckling to adult masticatory function. With advancing age, the lamina increased in thickness from 0.3 to 1.0 micron in adulthood, and was well demarcated from the subsurface tissue. The lamina was a continuous, smooth, electron-dense layer, that reacted biochemically as proteoglycan complex. The surface lamina may be responsible for the low frictional properties associated with articular movement and load, and the selective passage of molecules bidirectionally across the cartilage-fluid interface. Variations in lamina thickness occurred topographically across the disc and were attributed to site concentration of shearing stress. Beneath the articular surface lamina were cells surrounded by collagenous intercellular stroma. Chondrocytic, fibrocytic and fibrochondrocytic cellular phenotypes were present. Subsurface cellular debris accumulated within the rabbit disc from the commencement of mastication. Mature adult disc contained expanses of debris, attributed to cell death in situ. Foci of mineral precipitates were found within ageing discal tissue.

The effects of mandibular hypofunction on the development of the mandibular disc in the rabbit.

The effect of a reduced functional dentition on the development of the mandibular disc in young rabbits was studied by measuring cell proliferation within the disc following tooth extraction. Maxillary and mandibular incisor teeth were extracted from 18 animals at 5 weeks of age. At 12 weeks the rabbits received 0.25 mg/kg vincristine sulphate. Groups of three animals were killed 1.5, 3, 6, 12, 24 and 48 h after the injection of vincristine and the mitotic rate determined across the anterior, intermediate and posterior bands of the disc. Eighteen age- and sex-matched control rabbits with intact dentitions were treated in parallel. In the absence of incisor teeth, reflex gnawing and incising failed to develop, resulting in altered jaw movements and muscle force requirements. The mitotic rate in the anterior band was reduced significantly (p = 0.0117); rates for the intermediate and posterior bands were not significantly affected. There was an associated reduction in alveolar bone mass and deformation of the developing craniomandibular complex. As the lateral pterygoid inserts into the anterior band of the mandibular disc, it is proposed that altered activity within this muscle, combined with a modified loading of the joints, both secondary to incisor removal, resulted in a reduced mitotic rate in the anterior band of the developing mandibular disc.

The structure, innervation and location of arteriovenous anastomoses in the equine foot.

In the foot of the horse, arteriovenous anastomoses (AVAs) of epithelioid type occurred in the dermis of the coronary band, in the coronary and terminal papillae, in neurovascular bundles and at the entrance to and along the length of the dermal laminae. A particular feature of the epithelioid segment of AVAs in the horse, compared with that of other species, was the height and surface complexity of many of the endothelial cells. They extended into the lumen, forming undercut and tunnel-like areas which correlated with the characteristic surface marking of AVAs observed in vascular casts. The number of cell organelles, including the concentration of vesicles in the luminal cytoplasm, suggested cells with a high metabolic activity. The luminal surface possessed numerous microvilli and long cytoplasmic cell processes which appeared to surround material in the lumen. The innervation of AVAs was more dense than that of the arteries and consisted of adrenergic and peptidergic nerves. Noradrenaline- and neuropeptide Y-containing nerves were identified as the vasoconstrictor components of the nerve supply and occurred along arteries and formed dense plexuses around AVAs. Calcitonin gene-related peptide, substance P and vasoactive intestinal polypeptide are vasodilators and were present in single nerve fibres which accompanied arteries and AVAs along the length of the dermal laminae. In this study the distribution, density and innervation of AVAs in the equine foot are correlated with their proposed role in the development of acute laminitis. The release of vasoactive peptides from diseased organs remote from the foot may induce inappropriate prolonged dilatation of AVAs and thus contribute to the laminar ischaemia of acute laminitis.

The effects of induced dental malocclusion on the fibrocartilage disc of the adult rabbit temporomandibular joint.

Unilateral cast occlusal splints were fitted to the mandibular posterior teeth of adult rabbits, for periods of 1-28 days. The reactivity of the mandibular disc was examined by the effect on cell proliferation across the anterior, intermediate and posterior discal bands, as measured by metaphase arrest using vincristine sulphate. The effect on the disc was to activate cell proliferation on the splinted side. Intensity of response varied according to the length of time after fitting the splint, and the site involved. The findings suggested that the adult mandibular disc may participate in compensatory change at a cellular level and thus respond to changing functional loads placed upon the adult temporomandibular joints.

A scanning electron microscopical study of the dermal microcirculation of the equine foot.

The microcirculation of the dermal laminae and papillae of the equine foot from seven clinically normal Australian ponies was studied using an improved microvascular casting corrosion technique and scanning electron microscopy. Casts of veins, arteries, capillaries and arteriovenous anastomoses (AVAs) were readily identified by their characteristic surface morphology. Arteries entered the laminar circulation axially, between pairs of axial veins, and were connected to each other by smaller calibre interconnecting arteries. Short abaxial branches of the axial interconnecting arteries gave rise to tufts of predominantly, proximodistally orientated, capillaries arranged abaxially in rows. The laminar veins anastomosed with each other extensively (the axial venous plexus) and formed most of the vascular skeleton of casts of the dermal laminae. AVAs were found throughout the laminar circulation but the largest and longest (40 mu diameter) were found clustered close to the origin of the axial arteries. The density of the laminar AVAs was estimated to be 500 AVAs/cm2. Blood vessels of the dermal papillae of the periople, coronary band, distal laminae, sole and frog shared a basic structural organisation. The cast of each papillary unit consisted of a central artery and vein enmeshed in a sheath of fine capillaries. At intervals along the length of the central artery were short branches which gave rise to tufts of capillaries. The capillaries formed a tortuous anastomosing plexus which encircled the papillary unit and drained into the central vein at intervals along its length. AVAs were always present at the base of the papillary units and anastomoses connected the central artery and vein. AVAs are important components of the dermal microcirculation of the equine foot and their distribution and density is compatible with their proposed role in the pathophysiology of equine laminitis.

Vascular changes following hypothermic preservation.

This study identifies ultrastructural changes in the endothelium due to hypothermic HP-5 perfusion. These changes appear to be reversible and a manifestation of hypothermic hypoxia. This data provides a basis for improving perfusion techniques in both hypothermic preservation and cryoprotectant introduction and removal for cryopreservation.

Innervation of arteriovenous anastomoses in the sheep tongue: immunocytochemical evidence for coexistence of neural transmitters.

In this study structural and immunocytochemical evidence has shown that arterial vessels, particularly AVAs, are associated with nerves containing peptidergic vasodilators, viz. VIP, CGRP and SP. The presence of VIP-like immunoreactivity in both P-type and C-type nerves is evidence of the coexistence of VIP and acetylcholine in cholinergic nerves and suggests the action of VIP in maintaining the opening of AVAs in heat stress conditions. The evidence for the co-existence of CGRP and SP is more direct as immunoreactivity for both peptides has been demonstrated in serial sections of the same nerve terminal. Although SP is a potent vasodilator there is little evidence of its role in thermoregulation; however it may be involved in a local axon reflex and cause antidromic vasodilatation of local vessels particularly AVAs.

Innervation of arteriovenous anastomoses in the web of the foot of the domestic duck, Anas platyrhynchos: structural evidence for the presence of non-adrenergic non-cholinergic nerves.

An analysis of the innervation of arteriovenous anastomoses in the web of the foot of the Pekin Duck (Anas platyrhynchos) was compared with the innervation of the right atrium of the duck heart using histochemical, ultrastructural and morphometric techniques, before and after 6OHDA. The presence of intense catecholamine fluorescence and nerve terminals containing typical noradrenergic small dense-cored vesicles, together with the absence of fluorescence and degeneration of noradrenergic terminals after 6OHDA, indicated the presence of a dense adrenergic innervation at the periphery of the anastomoses. Ultrastructural and histochemical data gave support to the presence of a cholinergic innervation. There was evidence that arteriovenous anastomoses were innervated by non-adrenergic, non-cholinergic nerves, viz. after 6OHDA, the mean diameter, mean percentage and mean density of granular vesicles in axon profiles associated with anastomoses (107 . 25 nm, 22 . 34% and 12 . 73 vesicles micron-2, respectively) were significantly higher (P less than 0 . 001) than values in the atrium (87 . 13 nm, 9 . 92% and 5 . 51 vesicles micron-2, respectively) and axons associated with anastomoses contained large granular vesicles ranging up to 210 nm in diameter. This non-adrenergic non-cholinergic innervation may represent the non-adrenergic non-cholinergic vasodilatory nerves shown by pharmacological methods to be present in the foot of the Pekin Duck.

The use of ultrastructural and histochemical techniques to correlate sympathetic activity with blood flow through cutaneous arteriovenous anastomoses in conscious sheep.

In conscious, chronically prepared sheep, blood flow through cutaneous arteriovenous anastomoses (AVAs) was varied by manipulating spinal cord temperature. Ultrastructural examination of nerve terminals or varicosities adjacent to the media of the AVA wall showed that they contained numerous small clear or dense-cored vesicles typical of noradrenergic vesicles in sympathetic nerves. During spinal cooling about 1% of femoral flow passed through AVAs, the AVAs showed more intense catecholamine fluorescence and the percentage of dense-cored vesicles and their density was significantly greater than during spinal warming; up to 50% of femoral flow passed through AVAs during spinal warming. Our results conform with pharmacological and electrophysiological evidence for alpha-adrenergic involvement in control of AVAs. This correlation between structure and function could provide a useful method for (a) obtaining relative indices of blood flow through AVAs in body regions where techniques to measure blood flow cannot be applied, and (b) studying sympathetic control of cutaneous AVAs in experimental conditions under which electrophysiological techniques cannot be applied.

Arteriovenous anastomoses in the skin of seals. I. The Weddell seal Leptonychotes weddelli and the elephant seal Mirounga leonina (Pinnipedia: Phocidae).

The structure, distribution, density and innervation of arteriovenous anastomoses (AVAs) have been compared in the skin of Weddell and elephant seals, as part of a study of the structure and function of arteriovenous anastomoses in mammals. In both genera AVAs were coiled vessels with the segmental structure typical of "epithelioid" anastomoses and possessed a dense peripheral adrenergic innervation. In both Weddell and elephant seals there was no statistically significant difference between the mean density of AVAs in body skin and flipper skin. The majority, approximately 71% of AVAs occurred superficially in the dermis, fewer (23%) occurred in the deeper dermis, and 6% were present in the thick hypodermis (bubber). The density of AVAs in Weddell and elephant seals is approximately eight times greater than that reported in other animals and the superficial position of anastomoses over the whole of the body surface is characteristic of phocid seals. When open, AVAs in Weddell and elephant seals allow maximal heat loss from the skin surface by passing large volumes of blood into the superficial veins. AVAs in seals are important in dissipation of body heat, particularly when the animals are out of the water, and the entire surface area is thermoregulatory rather than specific regions such as the flippers.

Arteriovenous anastomoses in the skin of seals. II. The California sea lion Zalophus californianus and the northern fur seal Callorhinus ursinus (Pinnipedia: Otariidae).

The structure, distribution and density of arteriovenous anastomoses (AVAs) were studied in body and flipper skin of a California sea lion and a nothern fur seal. In both animals AVAs consisted of arterial, intermediate and venous segments, and were generally larger and more tortuous in the sea lion than in the fur seal. In the sea lion the majority of AVAs (72%) occurred in the deeper region of the dermis, and the density was significantly greater in the flippers than in the body. In the northern fur seal most AVAs (76%) occurred in the superficial region of the dermis; the density of AVAs in flipper skin was significantly higher than in body skin, and the density in the hind flipper was significantly greater than in the foreflipper. Arteriovenous anastomoses are important in the regulation of body temperature in seals; when these animals are on land, AVAs function to dissipate body heat, and vascular thermoregulation occurs in the flippers but notover the general body surface. Due to differences in distribution and density, AVAs play a more significant role in thermoregulation in the northern fur seal than in the California sea lion.