Influence of high temperature stress of 16-day embryos on subsequent hatchability

Two experiments were conducted to determine the effect of acute heat stress on late-stage chicken embryos. Embryos were incubated at a normal control temperature (37.5 degrees C.) for 16 days and were then subjected to 40.6, 43.3, 46.1 or 48.9 degrees C. for various periods of time in another incubator of the same type. At the end of the stress period all embryos were placed back into the control incubator for the remainder of the incubation period. Exposure of embryos for 24 hours to a temperature of 40.6 degrees C, caused no major detrimental effects on hatchability. Exposure for 6 hours to the temperature of 43.3 degrees C, caused a decrease in hatchability with a severe decline in hatchability occurring after 9 hours of exposure. Exposure to 46.1 degrees C. for 3 hours or 48.9 degrees C for 1 hour killed all embryos. Chicks which hatched following a severe heat stress had a high incidence of clubbed, wiry down and exhibited an unsteady gait.     

Maternal antioxidant treatments prevent diabetes-induced alterations of mitochondrial morphology in rat embryos

OBJECTIVE: To investigate the bioavailability (extent and rate of absorption) of ketorolac from two cutaneous absorption sources, active electrotransport and passive transdermal, and to examine the enantiomeric selectivity of bioavailability for each source. METHODS: Based on a crossover study in 12 healthy volunteers, the extent and rate of absorption of ketorolac, delivered by a patch, were found by estimating the input rate function of the drug. For that purpose, deconvolution was used in two steps. First, intravenous data were analyzed to estimate the ketorolac disposition function, and second, postpatch data were deconvolved to estimate the unknown patch input profile given the disposition function estimated in the first step. Because the input rate function curves to be estimated for the patches may be of arbitrary shape, a spline was used to model the patch input function, whereas intravenous data were modeled with use of a sum of exponentials. Differences in the extent of absorption (F) for the four treatment-enantiomer combinations were further examined with a mixed-effect regression model, based on the sets of four individual estimates of bioavailability. RESULTS: On average, the F value for the active electrotransport treatment, which exhibited the faster absorption rate, was four times greater than the F for the passive transdermal treatment. Further, during the passive treatment, R-ketorolac yielded an average F that is 42% greater than that for S-ketorolac and also exhibited a smaller absorption lag-time. During the active treatment, there was no important enantiomeric difference in either extent or rate of absorption.     

Heat-induced alterations in embryonic cytoskeletal and stress proteins precede somite malformations in rat embryos

Previous work from this laboratory has demonstrated that heat exposure on gestation day 10 (GD10) resulted in disrupted somite development 24 hr after exposure and subsequent thoracic skeletal malformations in neonates. The purpose of the present study was to examine the effects of in vitro heat shock on de novo protein synthesis and on cytoskeletal protein levels in developing rat embryos. Explanted GD10 embryos were exposed to temperatures of 42-42.5 degrees C for 15 min. At various times postexposure (0-27 hr). embryos were labeled with 35S-methionine and processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation. Transient enhanced de novo synthesis of 70- and 90-kD proteins was observed 1-8 hr after exposure. The 70-kD protein was identified as a eukaryotic stress protein and the presence of this protein was detected between 2 and 27 hr posttreatment. Western blot analysis was used to detect quantitative changes in total actin (microfilaments), tubulin (microtubules), and vimentin (intermediate filaments). Immediately following exposure, a reduction of total vimentin to minimal detectable levels was observed in heat-treated embryos. Levels of total vimentin remained depressed for more than 2 hr and gradually returned to control levels 4-8 hr postexposure. No change in total actin or tubulin was detected in treated embryos. The data demonstrate that heat-induced alterations in proteins comprising intermediate filaments occur concomitantly with the induction of stress proteins and precede aberrant somite morphology. These alterations in embryonic proteins may help elucidate the mechanism(s) by which skeletal malformations are produced.     

High frequency of p16INK4A gene alterations in hepatocellular carcinoma

The tumor suppressor gene p16 (CDKN2/MTS-1/INK4A) is an important component of the cell cycle and inactivation of the gene has been found in a variety of human cancers. In order to investigate the role of p16 gene in the tumorigenesis of hepatocellular carcinoma (HCC), 48 cases of HCC were analysed for p16 alterations by: methylation-specific PCR (MSP) to determine the methylation status of the p16 promoter region; comparative multiplex PCR to detect homozygous deletion; PCR-SSCP and DNA sequencing analysis to identify mutation of the p16 gene. We found high frequency of hypermethylation of the 5' CpG island of the p16 gene in 30 of 48 cases (62.5%) of HCC tumors. Moreover, homozygous deletion at p16 region were present in five of 48 cases (10.4%); and missense mutation were detected in three of 48 cases (6.3%). The overall frequency of p16 alterations, including homozygous deletion, mutation and hypermethylation, in HCC tumors was 70.8% (34 of 48 cases). These findings suggest that: (a) the inactivation of the p16 is a frequent event in HCC; (b) the p16 gene is inactivated by multiple mechanisms including homozygous deletion, promoter hypermethylation and point mutation; (c) the most common somatic alteration of the p16 gene in HCC is de novo hypermethylation of the 5' CpG island; and (d) in contrast to other studies, high frequency of genomic alterations are not uncommon in the 9p21 of the p16 gene. Our results strongly suggest that the p16 gene plays an important role in the pathogenesis of HCC.     

Release of luteinizing hormone-releasing hormone from enzymatically dispersed rat hypothalamic explants is pulsatile

Impaired whole blood fibrinolytic activity (FA), measured by the dilute clot lysis time (DCLT), is associated with first episodes of ischaemic heart disease (IHD) in the Northwick Park Heart Study in men, especially under 55 years, and in women. In a community-based study to investigate possible determinants of the DCLT, and therefore to assess which fibrinolytic components might be predictors of first IHD events, we measured fibrinolytic variables in a sub-sample of 150 healthy adults (73 males, 77 females) randomly selected from a single general practice. Most of the variance in DCLT (68% in men, 63% in women) was explained by tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) activities. In multiple regression analysis there was a significant difference in the strength of the association of t-PA activity with DCLT in men compared to women (test for interaction p = 0.05), the association of t-PA activity with DCLT being significant in males but not in females. Plasma PAI-1 activity was strongly associated with DCLT in both sexes. There was no independent association of DCLT with plasma fibrinogen, t-PA antigen, other fibrinolytic inhibitors, body mass index, serum lipids or C-reactive protein. Plasma PAI-1 activity in females and both t-PA and PAI-1 activities in males are the main determinants of whole blood FA measured by DCLT. It is therefore likely that these modulators of the plasma fibrinolytic system are associated with the onset of first clinical episodes of IHD. Elevated levels of t-PA antigen were positively associated with DCLT after adjustment for age and sex and therefore indicate impaired rather than enhanced FA. Further studies of the association of FA with risk of IHD should include not only "global" measures but also assessment of t-PA and PAI-1 activities, particularly as our results suggest that their associations with IHD may differ in men and women.     

Prostaglandin E2 and bacterial lipopolysaccharide stimulate bioactive interleukin-1 release from rat hypothalamic explants

While interleukin-1 (IL-1) is intimately involved in locally modulating the acute inflammatory response, it is also able to influence processes at remote sites, i.e., in an endocrine manner. While there is as yet little evidence that IL-1 can cross the blood-brain barrier, many effects such as fever, increased slow-wave sleep, anorexia and the modulation of neuroendocrine function suggest an action of circulating IL-1 at regulatory sites within the hypothalamus. However, there is accumulating evidence for IL-1 originating within the central nervous system (CNS), and it is currently unclear as to whether the neurally mediated manifestations of the acute inflammatory response are due to activation of central or peripheral (circulating) IL-1. In this study we have characterized the release of IL-1 from rat hypothalamic explants, and we have investigated the effects of putative modulators of IL-1 release, lipopolysaccharide (LPS) and the prostaglandins E2 (PGE2) and F2 alpha (PGF2 alpha). After 1 h of incubation, IL-1-like activity in hypothalamic supernatants ranged between 175 and 2,304 munits/mg of protein; this was substantially inhibited by the addition to the bioassay system of antibodies (1:200) against IL-1 alpha, but not against IL-1 beta. LPS and PGE2 significantly stimulated IL-1 release at 100 and 1 ng/ml respectively, whereas PGF2 alpha had no effect in the range of doses tested. It is therefore concluded that the control of hypothalamic IL-1 release may be investigated by means of acute rat hypothalamic explants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Constitutional alterations in p16 in patients with uveal melanoma

Chromosome 9p21 contains a susceptibility gene for cutaneous melanoma. Recent studies suggest that the gene responsible may be CDK41, since it encodes a putative cell cycle inhibitor, p16, and is frequently lost or rearranged in melanoma cell lines. In this study we examined whether germline alterations in CDK41 could be identified in patients with melanoma of the uveal tract. From an archive of bloods collected from patients with uveal melanoma, we identified 13 samples drawn from patients with a history in a family member of uveal (n = 6) or cutaneous (n = 7) melanoma. An additional 24 'control' bloods (without melanoma or any other primary malignancy in a family member), similar to the 'cases' in age and number of first-degree relatives, were also selected for study. For each sample, DNA was extracted from the red blood cell fraction. Using the polymerase chain reaction-single strand conformation polymorphism method, we screened for alterations in p16. Specific changes were characterized by DNA sequencing. Six nucleotide changes were detected in five (13.5%) of the 37 samples examined. An altered gene was found in one (7.7%) of the 13 patients with a family history (of intra-ocular melanoma) and four (16.7%) of the 24 patients with no family history (P = 0.64) of melanoma. In this series the group with a positive family history was predominantly female and most pedigrees involved matrilineal descent. In these data prevalence of germline alteration in p16 was similar in familial and sporadic cases. The results provide evidence against a significant role for p16 in familial clustering of intra-ocular and cutaneous melanomas.     

Attachment to the nuclear matrix mediates specific alterations in chromatin structure

The DNA in eukaryotic chromosomes is organized into a series of loops that are permanently attached at their bases to the nuclear scaffold or matrix at sequences known as scaffold-attachment or matrix-attachment regions. At present, it is not clear what effect affixation to the nuclear matrix has on chromatin architecture in important regulatory regions such as origins of replication or the promoter regions of genes. In the present study, we have investigated cell-cycle-dependent changes in the chromatin structure of a well characterized replication initiation zone in the amplified dihydrofolate reductase domain of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Replication can initiate at any of multiple potential sites scattered throughout the 55-kilobase intergenic region in this domain, with two subregions (termed ori-beta and ori-gamma) being somewhat preferred. We show here that the chromatin in the ori-beta and ori-gamma regions undergoes dramatic alterations in micrococcal nuclease hypersensitivity as cells cross the G1/S boundary, but only in those copies of the amplicon that are affixed to the nuclear matrix. In contrast, the fine structure of chromatin in the promoter of the dihydrofolate reductase gene does not change detectably as a function of matrix attachment or cell-cycle position. We suggest that attachment of DNA to the nuclear matrix plays an important role in modulating chromatin architecture, and this could facilitate the activity of origins of replication.     

Effects of injurious compression on matrix turnover around individual cells in calf articular cartilage explants

The effects of mechanical injury on the metabolism of cartilage matrix are of interest for understanding the pathogenesis of osteoarthrosis and the development of strategies for cartilage repair. The purpose of the present study was to examine the effects of injury on matrix turnover in a calf articular cartilage explant system for which the effects of mechanical loading on cell activity and the cell-mediated pathways of matrix metabolism are already well characterized. New methods of quantitative autoradiography were used in combination with established biochemical and biomechanical techniques for the analysis of cell and matrix responses to acute mechanical injury, with particular attention to the processes of localized matrix turnover in the cell-associated matrices of individual chondrocytes. Matrix deposition and turnover around cells in control explants was spatially dependent, with the highest rates of proteoglycan deposition and turnover and the lowest rates of collagen deposition (as indicated by [3H]proline autoradiography) occurring in the pericellular matrix. Injurious compression was associated with (a) an abrupt decrease in the tensile load-carrying capacity of the collagen matrix, apparently associated with mechanical failure of the tissue, (b) a considerable but subtotal decrease in cell viability, marked by the emergence of an apparently inactive cell population interspersed within catabolically active but abnormally large cells, and (c) sustained, elevated rates of proteoglycan turnover, particularly in the cell-associated matrices of apparently viable cells, which involved the increased release of aggregating species in addition to a spectrum of degradation fragments that were also in controls. These results may represent an in vitro model for the responses of chondrocytes and the cartilage extracellular matrix to mechanical injury.     

Analysis of heart development in cultured rat embryos

The long-range goal of this research is to establish an in vitro system that will permit pertubation of mammalian heart development and in situ examination of the cellular and molecular events underlying cardiac morphogenesis. Rat embryos at 9.5-11.5 days of gestation were placed in culture bottles containing rat serum and Tyrode's solution. Embryos cultured for 24 and 48 h were compared to age-matched in vivo controls for morphological score, morphometric analysis of heart development, and confocal and electron microscopic analysis of myofiber pattern formation. Morphological scores indicated that embryos cultured for 24 h from day 9.5 to 10.5 had essentially normal development when compared to age-matched embryos allowed to develop in vivo. Development of embryos maintained for 48 h in culture was slightly delayed at 66-68% of age matched in vivo embryos. Analysis of hearts from embryos allowed to develop 9.5-11.5 days in vivo plus 24 and 48 h in culture showed that the ventricular thickness and height, as well as the truncal, atrial and ventricular diameters were equivalent to those of hearts from age-matched in vivo controls. Hearts from embryos allowed to develop from 11.5-12.5 days in vitro and cultured for 24 and 48 h had smaller left ventricular and atrial dimensions than controls. Cardiac myofibrillogenesis and myofibrillar pattern formation in embryos cultured from 9.5 days of in vivo development for 48 h were also normal. These studies indicate that the rat whole embryo culture system is a useful model to study several critical periods in mammalian heart development.     

Age-related alterations in pre-synaptic and receptor-mediated cholinergic functions in rat brain

Fractional [3H]acetylcholine (ACh) release and regulation of release process by muscarinic receptors were studied in corpus striatum of young and aged rat brains. [3H] Quinuclidinyl benzilate (QNB) binding and carbachol stimulated phosphoinositide turnover, on the other hand, were compared in striatal, hippocampal and cortical tissues. High potassium (10 mM)-induced fractional [3H]ACh release from striatal slices was reduced by aging. Although inhibition of acetylcholinesterase with eserine (20 microM) significantly decreased stimulation-induced fractional [3H]ACh release in two groups of rats, this inhibition slightly lessened with aging. Incubation of striatal slices with muscarinic antagonists reversed eserine-induced inhibition in fractional [3H]ACh release with a similar order of potency (atropine = 4-DAMP > AF-DX 116 > pirenzepine) in young and aged rat striatum, but age-induced difference in stimulated ACh release was not abolish by muscarinic antagonists. These results suggested that fractional [3H]ACh release from striatum of both age groups is modulated mainly by M3 muscarinic receptor subtype. Although both muscarinic receptor density and labeling of inositol lipids with [myo-3H]inositol decreased with aging, carbachol-stimulated [3H]myo inositol-1-fosfat (IP1) accumulation was found similar in striatal, cortical and hippocampal slices.     

Gallium-67 scanning in the differentiation of maxillary sinus carcinoma from chronic maxillary sinusitis

Gallium-67 scans of 25 patients in whom the clinical symptoms and radiographic findings were suggestive of either maxillary sinus carcinoma or chronic sinusitis proved to be valuable in the differentiation between the two disease processes. Those patients with carcinoma had positive scans, while those with sinusitis had either negative or only weakly positive scans.     

Common nuclear matrix proteins in rat tissues

Nuclear matrix proteins have been defined as insoluble residual proteins resulting from treatment of isolated nuclei with nucleases, detergents and high ionic strength buffers. They are considered as in part representing the proteins constituting the three-dimensional framework of the interphase nucleus. Though cell-specific nuclear matrix proteins have been differentiated from ubiquitously occurring (common) nuclear matrix proteins, the number and types of common nuclear matrix proteins have not yet been unequivocally established. In the present study nuclear matrix proteins were prepared from isolated nuclei of rat kidney, liver, lung, spleen and testes. The matrix proteins were separated by two-dimensional (2-D) electrophoresis and silver stained. Then the spot patterns were compared by computer-assisted image analysis. Composite images were derived for nuclear matrix proteins of individual tissues. Finding between 396-483 spots per tissue, a total of 964 individual spots were registered. Of these, 102 were common nuclear matrix proteins, as appearing in each of the tissue-characteristic images. The apparent molecular mass and pI data may serve for further identification of these nuclear proteins.     

Three-dimensional reconstructions of the developing forebrain in rat embryos

Using a computerized three-dimensional reconstruction technique with serially sectioned rat embryos, changes in the size and form of the forebrain were studied on Embryonic Days (E) 12 (1 day after closure of the neural tube), E15, E18, and E21 (2 days before birth). During this time, the forebrain changes from a relatively simple tubular structure with thin walls surrounding a large ventricular system to a thick-walled brain with a highly convoluted but reduced ventricular system. On E12, the two components of the forebrain, the telencephalon and the diencephalon, cannot be distinguished. Considering the forebrain as a whole (the embryonic prosencephalon), its volume continually increases between E12 and E21 due to the generation, differentiation, and maturation of neurons and glia. Attention was paid to changes in the sizes of the ventricles, the neuroepithelium and the parenchyma. Volumes of the ventricles and the surrounding neuroepithelium rapidly expanded from E12 to E18 and then decreased by E21, while the volume of the parenchyma continually increased. Differential growth of the telencephalon and that of the diencephalon were compared between E15 and E21. The expansion of the telencephalon was much larger than that of the diencephalon. In the telencephalon, the volumes of the lateral ventricles and the surrounding neuroepithelium increased between E15 and E18 and decreased by E21, while in the diencephalon the volumes of the third ventricle and its surrounding neuroepithelium continually declined between E15 and E21. That observation is compatible with previous work showing that the majority of diencephalic structures develop earlier than those in the telencephalon. It is important to note that volume changes in the ventricles and the neuroepithelium are maintained in "lock-step," suggesting a close relationship between the size of the ventricle and the size of the neuroepithelium.     

Cannabinoid-induced alterations in regional cerebral blood flow in the rat

A specific receptor for cannabinoids has been characterized at the pharmacological, molecular, and neuroanatomical level. However, less is known of the functional localization in the brain for the behavioral and physiological actions of these drugs. We have examined the effects of delta 9-tetrahydrocannabinol (THC) and its active metabolite 11-OH-THC on regional cerebral blood flow in the rat in order to determine functional CNS sites of action for the cannabinoids. Conscious rats were injected i.v. with one of four doses of THC (0.5, 1, 4, 16 mg/kg). 11-OH-THC (4 mg/kg), or vehicle 30 min prior to sacrifice. Regional cerebral blood flow was determined autoradiographically using the freely diffusible tracer method of Sakaruda et al. Changes in regional cerebral blood flow were observed in 16 of the 37 areas measured. Decreases in regional cerebral blood flow following THC were seen in such areas as the CA1 region of the hippocampus, frontal and medial prefrontal cortex, the nucleus accumbens, and the claustrum. Thresholds for these effects ranged from 0.5 to 16 mg/kg. Areas unaffected by THC include the medial septum, ventral tegmental area, caudate, temporal, parietal and occipital cortex, and cerebellum. These data indicate that THC and its active metabolite, 11-OH-THC, cause a heterogeneous alteration in the activity of specific CNS sites, many of which are involved in the characteristic behavioral actions of THC.     

Golgi complex alterations induced by X537A in chief cells of rat parathyroid gland

Electron microscopic examination of chief cells of rat parathyroid glands incubated with the antibiotic ionophore X537A showed selective alterations of the Golgi complex. The changes were dependent on the duration of X537A exposure. After 50 minutes of incubation, the cisternae and vesicles of the Golgi complex appeared highly swollen and disorganized. Because of its selective action, X537A might be a useful tool in the investigation of the role of Golgi apparatus in parathormone secretion.     

A similar pattern of chromosomal alterations in prostate cancers from African-Americans and Caucasian Americans

A combination of genetic and epigenetic factors may explain the disproportionate incidence and mortality of prostate cancer among African-American males (AAMs) as compared with Caucasian American males (CAMs). We wished to determine whether primary prostate cancers from AAMs and CAMs harbor different patterns or frequencies of chromosomal alterations. Comparative genomic hybridization (CGH) was performed on clinically localized, untreated primary prostate cancers from 16 AAMs and 16 CAMs. Detailed statistical analysis was used to delineate gains and deletions with high sensitivity and specificity and to compare the frequency and pattern of alterations between the two groups of tumors. The two groups of patients had indistinguishable preoperative serum prostate-specific antigen levels, and the two groups of tumors had similar pathological stages and grades. Chromosomal gains and deletions occurred in regions known to be frequently altered in prostate cancer. Specifically, the most frequent alterations were deletions of regions on chromosomes 13q, 5q, 16q, and 8p and gains of regions on 8q and 5q. When tumors from AAMs and CAMs were compared, the frequencies of alteration (deletion, gain, or no alteration) were similar across 98.9% of the length of the genome. The patterns of alterations of the most frequently altered chromosomes were also similar between tumors from AAMs and CAMs. We concluded that primary prostate cancers from AAMs and CAMs harbor a similar pattern and frequency of chromosomal alterations. These data support the notion that sporadic prostate cancers from AAMs and CAMs develop by similar chromosomal mechanisms. Biological differences, if present, do not occur on the chromosomal level.     

Evidence that carbon monoxide stimulates prostaglandin endoperoxide synthase activity in rat hypothalamic explants and in primary cultures of rat hypothalamic astrocytes

Freezing or freeze-drying and gamma-irradiation are techniques currently used for processing tendon allografts. However, it is still unknown how these processing methods affect graft remodeling. In this study, we used a rat patellar tendon transplantation model to investigate the effect of various processing methods on remodeling by quantifying loss of collagen labeled with a radioactive isotope. The grafts were divided into the following four groups according to the processing method: fresh-frozen, freeze-dried, fresh-frozen and gamma-irradiated, or freeze-dried and gamma-irradiated. The percentage of donor collagen, calculated from hydroxyproline content and radioactivity level, was used as an indicator of graft remodeling. At 2 weeks, the level of donor collagen in the fresh-frozen group was 62%; in the freeze-dried group, 59%; in the fresh-frozen and irradiated group, 57%; and in the freeze-dried and irradiated group, 44%. At 4 weeks, the percentage of donor collagen remaining in grafts decreased to 38% in the fresh-frozen group, 19% in the freeze-dried group, 27% in the fresh-frozen and irradiated group, and 12% in the freeze-dried and irradiated group. Finally, at 12 weeks, the levels were 19% in the fresh-frozen group, 20% in the freeze-dried group, 15% in the fresh-frozen and irradiated group, and 6% in the freeze-dried and irradiated group. The percentages of donor collagen in the freeze-dried and the fresh-frozen and irradiated groups were significantly lower than that in the fresh-frozen group at 4 weeks. The values for the freeze-dried and irradiated group were significantly lower than those for the fresh-frozen and irradiated group at 4 and 12 weeks. These data suggest that freeze-drying, freeze-drying followed by gamma-irradiation, and fresh-freezing followed by gamma-irradiation temporarily accelerate graft remodeling.