Iron-stabilized carbocations as intermediates for organic synthesis.

Attachment of a transition metal moiety to an olefinic ligand presents the organic chemist with unequaled opportunities to control the regio- and stereospecificities of bond formation. Applications of cationic dienyliron-carbonyl complexes to a range of natural product syntheses have been developed. These applications show how the iron-carbonyl unit directs the regio- and stereochemistry of nucleophile addition. They also show that the iron-carbonyl unit can be used to stabilize otherwise inaccessible carbocations, thereby making them readily available as synthetic intermediates.

Prolactin delays hair regrowth in mice.

Mammalian hair growth is cyclic, with hair-producing follicles alternating between active (anagen) and quiescent (telogen) phases. The timing of hair cycles is advanced in prolactin receptor (PRLR) knockout mice, suggesting that prolactin has a role in regulating follicle cycling. In this study, the relationship between profiles of circulating prolactin and the first post-natal hair growth cycle was examined in female Balb/c mice. Prolactin was found to increase at 3 weeks of age, prior to the onset of anagen 1 week later. Expression of PRLR mRNA in skin increased fourfold during early anagen. This was followed by upregulation of prolactin mRNA, also expressed in the skin. Pharmacological suppression of pituitary prolactin advanced dorsal hair growth by 3.5 days. Normal hair cycling was restored by replacement with exogenous prolactin for 3 days. Increasing the duration of prolactin treatment further retarded entry into anagen. However, prolactin treatments, which began after follicles had entered anagen at 26 days of age, did not alter the subsequent progression of the hair cycle. Skin from PRLR-deficient mice grafted onto endocrine-normal hosts underwent more rapid hair cycling than comparable wild-type grafts, with reduced duration of the telogen phase. These experiments demonstrate that prolactin regulates the timing of hair growth cycles in mice via a direct effect on the skin, rather than solely via the modulation of other endocrine factors.

Wavelength dependence of cellular responses in human melanocytes and melanoma cells following exposure to ultraviolet radiation.

PURPOSE: To examine the wavelength dependence of cellular responses in human melanocytes and human melanoma cells exposed to ultraviolet radiation (UVR). MATERIALS AND METHODS: Primary human melanocytes and G361 human melanoma cells were exposed to ultraviolet-C (UVC), ultraviolet-B (UVB), or ultraviolet-A (UVA) radiation. Dose-response relationships for clonal cell survival were assessed, and flow cytometry was used to monitor cell cycle distributions for up to one week post-irradiation. Chromosomal aberrations were scored in exposed and unexposed melanoma cells. RESULTS: G361 melanoma cells were more sensitive than melanocytes to killing by UVB and UVC radiation. This difference in sensitivity between cell types was much less marked following UVA irradiation. The melanoma cells showed a sustained, dose-dependent G2/M block following exposure with all wavelengths; in addition, transit through S phase was slowed following UVA irradiation. There was no apparent block to G1 cells entering S phase at any wavelength. Melanocytes, on the other hand, showed a marked G1 arrest, particularly following UVA irradiation. Cytogenetic results showed a dose-dependent increase in chromatid-type aberrations, mostly gaps, breaks and exchanges, in exposed melanoma cells. CONCLUSION: These results show that G361 malignant melanoma cells have lost the ability to regulate the cell cycle at the G1/S checkpoint and are more sensitive than melanocytes to cell killing by UVC and UVB but not UVA radiation. Similarly, exposure of these melanoma cells to UVC and UVB, and to a much lesser extent UVA, induced chromatid aberrations. UVA nevertheless induced strong cell cycle delays in both cell types, indicating that UVA exposure can significantly affect genome metabolism.

Identification of differentially expressed genes during a wool follicle growth cycle induced by prolactin.

The wool follicles of New Zealand Wiltshire sheep can be induced to undergo growth cycles by manipulating circulating prolactin levels. Altered patterns of gene expression through this cycle were examined using differential display, and nine sequence tags for differentially expressed genes were isolated. Four of these tags were identified as fragments of known genes, encoding a wool keratin, KRTAP3.2, a desmosome component, desmoglein 1, an epithelial cell marker, stratifin, and a protein kinase, Clk3. All four genes were shown to be downregulated in telogen skin compared with anagen. In situ hybridization showed that all had localization patterns which included cells that are absent in telogen. The stratifin tag was used to clone a cDNA that incorporated a complete open-reading frame for ovine stratifin. Ovine stratifin is similar to the human form, showing only six single residue differences in the predicted amino acid sequence. Stratifin probably acts as a regulator of other proteins involved in trichocyte cell cycling and differentiation. Clk3 is involved in regulating RNA splicing. KRTAP3.2 and Dsg1 both play structural roles in hair follicles. The other five tags, including two representing genes that were upregulated during catagen, could not be identified by homology. Differential display is an effective means of identifying genes involved in follicle function and, potentially, of genes controlling the growth cycle.

Prolactin signaling influences the timing mechanism of the hair follicle: analysis of hair growth cycles in prolactin receptor knockout mice.

Pituitary PRL regulates seasonal hair follicle growth cycles in many mammals. Here we present the first evidence implicating PRL in the nonseasonal, wave-like pelage replacement of laboratory mice. In this study we show that messenger RNA transcripts encoding the one long and two short forms of PRL receptor are present in the skin of adult and neonate mice. The receptor protein was immunolocalized to the hair follicle as well as the epidermis and sebaceous glands. Furthermore, PRL messenger RNA was detected within skin extracts, suggesting a possible autocrine/paracrine role. Analysis of the hair growth phenotype of PRL gene-disrupted mice (PRLR(-/-)) revealed a change in the timing of hair cycling events. Although no hair follicle development differences were noted in PRLR(-/-) neonates, observations of the second generation of hair growth revealed PRLR(-/-) mice molted earlier than wild types (PRLR(+/+)). The advance was greater in females (29 days) than in males (4 days), resulting in the elimination of the sexual dimorphism associated with murine hair replacement. Heterozygotes were intermediate between PRLR(-/-) and PRLR(+/+) mice in molt onset. Once initiated, the pattern and progression of the molt across the body were similar in all genotypes. Although all fiber types were present and appeared structurally normal, PRLR(-/-) mice had slightly longer and coarser hair than wild types. These findings demonstrate that PRL has an inhibitory effect on murine hair cycle events. The pituitary PRL regulation of hair follicle cycles observed in seasonally responsive mammals may be a result of pituitary PRL interacting with a local regulatory mechanism.

Transforming growth factor-alpha immunoreactivity during induced hair follicle growth cycles in sheep and ferrets.

Transforming growth factor-alpha (TGF-alpha) has been associated with cell proliferation of keratinocytes and implicated in hair growth. We therefore examined changes in the immunocytochemical localization of TGF-alpha and cell proliferation markers in the skin of two unrelated species in which hair cycles could be induced, to elucidate the role of this growth factor in the control of fiber growth. Skin was collected from melatonin-treated ferrets (Mustela putorius furo), untreated Romney sheep (Ovis aries), and New Zealand Wiltshire sheep in which interruption of wool growth had been photoperiodically induced. Immunostaining patterns were very similar in ferrets and sheep. TGF-alpha immunoreactivity was observed in epithelial tissues of the skin but was not co-localized with cell proliferation markers. In anagen follicles, specific staining was most intense in the innermost cells of the outer root sheath and cortical cells in the keratogenous zone but was absent from inner root sheath or dermal papilla. TGF-alpha immunostaining diminished during catagen, although faint staining was retained in all epithelial cells. In telogen and early proanagen follicles, staining remained faint or was restricted to cells on the margin of the brush end and follicle neck. Immunoreactivity in the outer root sheath was reestablished in late proanagen. Sebaceous glands and epidermis were stained intensely throughout the hair cycle. TGF-alpha-immunoreactive components of skin extracts, analyzed by Western blotting, showed mobility corresponding to approximately 32 KD, but not to the size of the fully cleaved peptide. These results are consistent with an epithelial autocrine or juxtacrine, but not a mitogenic, function of TGF-alpha.

Distribution of prolactin receptor immunoreactivity in ovine skin and changes during the wool follicle growth cycle.

Prolactin is believed to mediate seasonal cues entraining seasonal reproductive and hair follicle growth cycles. Prolactin receptor binding activity and prolactin receptor gene expression in mammalian skin have recently been described. In this report, prolactin receptor immunoreactivity is identified in sheep skin using a monoclonal antibody against the rat liver prolactin receptor. Western blotting analysis of microsomal membrane proteins from skin showed major bands corresponding to molecular weights of 87 and 71 kDa and minor bands at 101 and 21 kDa. RNase protection analysis revealed the presence of mRNA species coding for long and short forms of the prolactin receptor. Formalin-fixed sections, exposed to the monoclonal antibody and stained by an immunogold method, revealed prolactin receptor-immunoreactivity in the dermal papilla, germinal matrix, outer root sheath, lower regions of the inner root sheath and connective tissue sheath of wool follicles. Staining was absent from keratinised cell populations. In all samples, the interfollicular epidermis, sebaceous and sweat glands were positively stained. The distribution of prolactin receptor is described in both growing and inactive wool follicles and related to postulated cycle-specific actions of circulating prolactin in the control of seasonal fibre growth.

Localisation of receptors for prolactin in ovine skin.

Although prolactin (PRL) receptors are found in many mammalian tissues, specific PRL binding to mammalian skin has not been demonstrated. In view of the temporal relationships observed between photoperiod, circulating PRL and pelage replacement in seasonally responsive mammals, we sought to provide evidence of PRL receptors in ovine skin. Cryosections of skin from New Zealand Wiltshire ewes were incubated with radiolabelled human GH (125I-hGH) and ovine PRL (125I-oPRL) in the presence and absence of excess unlabelled hormones (hGH, oPRL or ovine GH (oGH)). Binding was inhibited by unlabelled oPRL and hGH but not by oGH. In microautoradiographs, both radioligands were localised most strongly in the dermal papillae of wool follicles in the anagen (growth) phase of the hair cycle and in apocrine sweat glands. Higher levels of specific binding to dermal papilla cells, compared with the follicle epithelial matrix and the surrounding dermis, were confirmed by measurement of microautoradiograph silver grain density (respectively, 34.1 +/- 3.0, 11.4 +/- 1.0 and 5.5 +/- 0.5 grains per 100 microns2 (mean +/- S.E.M., n = 10)). Total binding for 125I-hGH and 125I-oPRL radioligands to follicle dermal papilla was not significantly different (34.1 +/- 3.0 vs 43.6 +/- 2.5 grains per 100 microns2, n = 10) but the level of non-specific binding of 123I-oPRL was higher than for 125I-hGH (18.9 +/- 1.4 vs 6.1 +/- 0.6 grains per 100 microns2, n = 10; P < 0.001). Binding assays of receptors in crude microsomal membranes extracted from ovine skin were used to ascertain binding capacity and specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effect of increased photoperiod on wool follicle growth.

The relationships between circulating prolactin (PRL), wool follicle growth and daylength were investigated in 24 New Zealand Wiltshire ewes housed indoors from September 1989 to May 1991. Twelve control (C) ewes were maintained under natural photoperiod. Two other groups were held in short days (SD; 8 h light: 16 h darkness) commencing from the winter solstice (22 June 1990) for either three (group SD3, n = 7) or six (group SD6, n = 5) months before reversion to natural daylength. Skin was sampled at one- to four-week intervals for histological determination of percentages of growing primary and secondary follicles. Hourly blood samples over 24 h were collected via jugular cannulae from C sheep in March and July and then monthly from all animals until December 1990 for estimation of mean monthly PRL concentrations for each treatment group. Between autumn (March 1990) and winter (July) primary follicle activity (PFA) and secondary follicle activity (SFA) declined in C ewes (PFA: 97 to 43%, SFA: 100 to 57%). Follicle regrowth during July and August in eight C ewes preceded the initial rise in plasma PRL from the winter minimum (1.6 ng/ml). Across the three groups, four instances of decreased follicle activity were observed, closely following or concurrent with increases in plasma PRL concentrations. The resumption of spring growth in four C sheep was temporarily checked by falls in follicle activities during September and October as PRL concentrations began to increase (3.4 to 8.9 ng/ml). Follicle activity also declined in November and December in eight C sheep, coincident with the rapid rise in PRL to a seasonal maximum in late November (165.4 ng/ml). The increase in SD3 follicle activity over spring was not delayed by short days but during October, after release from treatment, PRL concentrations rose (1.8 to 12.0 ng/ml) and follicle activity declined (PFA: 65 to 38%, SFA: 68 to 43%). In SD6 ewes, PRL concentrations were suppressed (2.1 ng/ml) and relatively constant levels of follicle activity (PFA: 73%, SFA: 95%) were maintained throughout short-day treatment. Release of SD6 ewes into summer photoperiod in January 1991 temporarily interrupted follicle growth (PFA: 68 to 17%, SFA: 96 to 19%) and caused out-of-season shedding in March and April. Contemporary C follicle activities were high (PFA: 95%, SFA: 98%). These data suggest that natural and experimental increases in daylength have a short-term inhibitory effect on growing wool follicles which could be mediated through rising concentrations of plasma prolactin.

Regulation of prolactin receptor expression in ovine skin in relation to circulating prolactin and wool follicle growth status.

Seasonal patterns of hair growth are governed, at least in part, by levels of prolactin in circulation, and although receptors for prolactin (PRLR) have been demonstrated in hair follicles, little is known of their regulation in relation to follicular cycles. In this study, a photoperiod-generated increase in prolactin was used to induce a wool follicle cycle during which changes in PRLR expression in sheep skin were determined by ribonuclease protection assay and in situ hybridisation. mRNA for prolactin and both isoforms of PRLR were also detected in skin by reverse transcription and polymerase chain reaction. As circulating prolactin began to rise from low levels, PRLR mRNA in the skin initially fell. These changes immediately preceded the catagen (regressive) phase of the hair cycle. Further increase in prolactin resulted in up-regulation of PRLR during telogen (dormancy), particularly in the epithelial hair germ, to reach a peak during proanagen (reactivation). In anagen (when follicle growth was fully re-established), PRLR mRNA returned to levels similar to those observed before the induced cycle. Hence, this longer term rise and fall of PRLR expression followed that of plasma prolactin concentration with a lag of 12-14 days. PRLR mRNA was most abundant in the dermal papilla, outer root sheath, hair germ, skin glands and epidermis. Location of PRLR in the dermal papilla and outer root sheath indicates action of prolactin on the growth-controlling centres within wool follicles. These cycle-related patterns of PRLR expression suggest dynamic regulation of PRLR by prolactin, thereby modulating hormonal responsiveness of seasonally growing hair follicles.

Skin explant culture as a model for cutaneous graft-versus-host disease in humans.

An in vitro skin explant model for graft-versus-host disease (GVHD) in humans has been used to study the role of effector T cells in the histological pathogenesis of GVHD. In 11 of 12 experiments clear GVHD changes of grades II-IV were induced in HLA-mismatched skin explants cultured with allogeneic T cells sensitized by in vitro mixed lymphocyte culture. The role of effector T cells was investigated by comparing results before and after removal of CD3 positive cells, and CD4 positive and CD8 positive T cell-subsets by antibody and complement cytolysis from responder populations. Only total removal of CD3 positive T cells prevented histopathological lesions of GVHD in the skin biopsy specimens. The results also demonstrated that the CD4 positive population caused the greatest degree of GVHD in vitro in skin biopsy specimens and direct infiltration into skin by cells is not required for changes to become evident. These results confirm the early results on animal models and demonstrate the use of the skin explant model as a tool for studying the biology of GVHD in humans.

Stereocontrolled construction of tetrahydrofurans and gamma-butyrolactones using organomolybdenum chemistry.

[reaction: see text]. Diastereoselective conversion of pi-allylmolybdenum complex aldehyde 1 to organometallic triol 4 and diols 5, 10, and 13 is described. Stereocontrolled demetalation of 4, 5, and 13 was accomplished, leading to hydroxylated tetrahydrofurans and gamma-butyrolactones, as single diastereoisomers.

Approaches to the fully functionalized DEF ring system of ristocetin A via highly selective ruthenium-promoted S(N)Ar reaction.

Ruthenium-promoted intramolecular S(N)Ar reaction has allowed the construction of the fully functionalized 16-membered DEF macrocycle 4 of ristocetin A that incorporates the required arylglycine and arylserine residues as the F and E ring, respectively.

The infusion requirements and recovery characteristics of cisatracurium or atracurium in intensive care patients.

OBJECTIVE: To investigate the infusion requirements and recovery characteristics of cisatracurium compared with atracurium when both are administered by prolonged continuous infusion. DESIGN: A prospective, randomised, single-blind study. SETTINGS: The Intensive Care Unit of the Manchester Royal Infirmary. PATIENTS: 20 patients requiring a continuous infusion of a neuromuscular blocking agent to facilitate mechanical ventilation. 12 patients received cisatracurium and 8 received atracurium. INTERVENTIONS: Cisatracurium or atracurium was administered by continuous infusion for a minimum of 24 h. The level of neuromuscular blockade was measured by recording the train-of-four responses using acceleromyography, the aim being to maintain 1-2 twitch responses of the adductor pollicis. At the end of the infusion period, the train-of-four was recorded until the ratio was greater than 0.7. MEASUREMENTS AND RESULTS: The mean infusion rate of cisatracurium was 0.23 mg kg-1 h-1, compared to 0.62 mg kg-1 h-1. No time-related increase in infusion requirements was seen for either drug. The mean recovery time to a train-of-four ratio greater than 0.7 was the same (46 min). There was no correlation between recovery time and age, duration of infusion or mean infusion rate. CONCLUSIONS: Cisatracurium provides a satisfactory level of neuromuscular blockade in adult ICU patients at approximately one-third the infusion rate of atracurium and with a similar recovery time.

Role of stress responses in human cell survival following exposure to ultraviolet C radiation.

PURPOSE: To investigate in human skin and other cells the role of tyrosine kinase and protein kinase-C (PKC) in eliciting cell-signalling responses to UV radiation (UVR) that affect the survival of irradiated cells. MATERIALS AND METHODS: The survival of HeLa S3 cells, NCTC 2544 human keratinocytes and A431 human epidermal carcinoma cells was measured following incubation with various tyrosine kinase or PKC inhibitors and exposure to UVC (254nm) radiation. In addition, Western blotting measured PKC isozyme expression in human keratinocytes following UVC exposure. RESULTS: It was confirmed that inhibition of tyrosine kinase activation reduces the survival of UV-irradiated HeLa S3 cells. However, no effect was seen on the survival of either NCTC 2544 human keratinocytes or A431 human epidermal carcinoma cells. In contrast, specific inhibition of PKC reduced the survival of UV-irradiated keratinocytes but had no effect on HeLa cells. Comparison of the effects of different inhibitors in keratinocytes suggested that this effect was mediated mostly through PKCmu and PKClambda/iota. In addition, keratinocyte exposure to UVC induced large and temporally distinct increases in PKCmu and PKClambda/iota. CONCLUSIONS: The survival of NCTC 2544 keratinocytes, but not HeLa S3 cells, following UVC exposure is mediated by signalling through PKC, mostly PKCmu and PKClambda/iota. Further study is required to confirm these results in normal human keratinocytes.

Carcass composition and meat quality of brush-tail possums (Trichosurus vulpecula).

One-hundred-and-thirty-nine possums, balanced as far as possible for sex (68 male, 71 female) and age (1 year, 42; 2-4 years, 61; 5+ years, 36), were slaughtered and the dissected carcass composition, muscle, and cooked meat composition measured. Possums were slaughtered at time in captivity TIC 0 (n = 58), TIC 14 days (n = 20) and TIC 28 days (n = 61). Compared with the 5+ years (mature) age group the 1 year olds (juveniles) were 67% and the 2-4 year olds (immature) 96% of the live weight of the mature possums. Carcass composition was characterised by high lean (78-80%) and low fat (around 1-2%). The cooked meat from possums is very high in protein (∼ 25%) and low in fat, suggesting it should be a valuable source of animal protein with a low total fat, high unsaturated fat content. In all cases cooked meat was rated as tender, based on shear force values, despite having a pH of around 6·3.

Transmission of Leptospira interrogans serovar Balcanica infection among socially housed brushtail possums in New Zealand.

Leptospira interrogans serovar balcanica is a potential vector being investigated for spreading a biological control agent among introduced brushtail possums (Trichosurus vulpecula) in New Zealand. As previous studies have shown that possums are unlikely to contract leptospirosis through a contaminated environment alone, the objective was to determine whether L. interrogans serovar balcanica could be transmitted between sexually mature, socially housed possums. Possums were infected experimentally with L. interrogans serovar balcanica and housed in pairs or groups with uninfected possums for either 70 or 140 days, during the breeding or non-breeding seasons. No transmission occurred between any infected and uninfected possums during the non-breeding season. However, transmission occurred between females that had been socially housed in pairs or groups in the breeding season. Mixed sex transmission also occurred in pairs and groups, both from males to females and from females to males. Mixed sex transmission usually occurred rapidly (< 44 days) and was not associated with the production of offspring. No transmission occurred between males during the breeding or the non-breeding seasons. Transmission probably occurs as a result of affiliative or sexual behaviour, but is unlikely to occur through fighting. The social transmission pathways determined in this study suggest that L. interrogans serovar balcanica may have the transmission attributes desired in a vector for biological control.

Leptospirosis in brushtail possums: is Leptospira interrogans serovar balcanica environmentally transmitted?

In New Zealand, the biological control of introduced brushtail possums (Trichosurus vulpecula) may be the only affordable option for achieving a significant long term reduction in pest numbers on a national scale. Leptospira interrogans serovar balcanica is among the potential biocontrol agents and vectors currently being investigated for this purpose. As the transmission pathways of L. interrogans serovar balcanica between possums are poorly understood, the objective of the study was to determine whether infection could result from exposure to contaminated environments. Sixteen individually housed, uninfected possums, in three groups, were regularly exposed over a period of 32 days to contaminated cages or grass enclosures of 16 other experimentally infected possums all shedding leptospires in their urine. None of the 16 challenged possums developed serological evidence of L. interrogans serovar balcancia infection. These results suggest that this organism is unlikely to be transmitted environmentally, supporting previous circumstantial evidence that social contact may be required for transmission of L. interrogans serovar balcanica between possums.

Psychoneuroimmunology and preterm birth. A holistic model for obstetrical nursing practice and research.

This article presents a framework through which nurses can conceptualize premature labor and birth for both practice and research. Use of the psychoneuroimmunology (PNI) model may guide the study of the problem of preterm birth in a more holistic manner, discovering relationships between the body and the mind that may affect how nurses can intervene to prevent premature birth. Nursing assessment of risk needs to include those situations that may lead to increased stress or anxiety, as is supported by research based on the PNI model. Reduction of stressors that lead to physiological changes related to the stress response can affect the incidence of preterm labor. Interventions to decrease stress and poor coping behaviors need to be tested and integrated into practice.

A novel zoometric index for assessing body composition in adult rabbits.

Obesity is an emerging problem in domesticated rabbits, and an easy-to-use measure of adipose tissue mass is needed. The current study aimed to develop a zoometric ratio, capable of estimating body condition in rabbits. Body weight (BW), body condition score (BCS), and zoometric measures (distal forelimb length, DFL; vertebral length, VL were measured in 150 pet rabbits. Zoometric formulae were created, combining BW with a zoometric measure, and these were tested for their ability to predict adipose tissue mass judged by BCS. Seventy-five (50 per cent) of the rabbits were in ideal condition (BCS 2.5-3.5), 52 (35 per cent) were overweight (BCS>3.5), and 23 (15 per cent) were underweight (BCS<2.5). Median (range) DFL and VL measurements were 12.1 (8.8-16.4 cm) and 34.0 (26.5-50.5 cm), respectively. In rabbits of medium breed size, the BW/DFL ratio was most strongly associated with BCS (Kendall's τ 0.80, P<0.001). Using BW/DFL limits for optimum body condition (eg, minimum 0.16; maximum 0.21), all underweight and overweight rabbits were correctly classified, while only 2/61 (3 per cent) rabbits with an optimum BCS were incorrectly classified as overweight. This study provides preliminary evidence that the BW/DFL might be a useful indirect measure of adipose tissue mass in rabbits of medium breed size.