Lack of correlation between thymidylate synthase levels in primary colorectal tumours and subsequent response to chemotherapy.

The increasing interest in 5-fluorouracil (5-FU) modulation and the development of new antifolates has focused attention in recent studies on the expression of the target enzyme thymidylate synthase (TS) as a determinant of drug sensitivity and resistance. Resistance to TS-directed drugs has been shown to occur in vitro and in vivo with increased expression of the enzyme (determined by enzymatic assays as well as protein and gene expression assays). Several studies have evaluated the role of TS as a prognostic indicator of clinical response to chemotherapy containing TS-directed drugs. We have used a polyclonal antibody to recombinant human TS to establish a silver-enhanced immunogold staining method to localize TS in human tumours. Human tumour cell lines with acquired resistance to TS inhibitors owing to increased levels of TS were used to confirm the specificity of immunostaining. Stained sections were evaluated by image analysis. Immunostaining in tumour sections was greatly reduced (>80%) by preabsorption of the antiserum with recombinant TS. The method was used to determine the extent of TS immunostaining in 134 primary human colorectal tumours. The results were then compared with the clinical outcome and response to chemotherapy for the treatment of subsequent metastatic disease. A wide range (approximately 100-fold) of TS immunostaining was observed in these primary tumour sections. Normal mucosal tissue levels were 5-10 times lower than those observed in the adjacent tumour tissue. The values for TS immunostaining did not correlate with clinical endpoints, such as time from diagnosis to relapse, response to chemotherapy for disseminated disease, nor with Dukes'' staging. This lack of correlation may be because this group of patients was selected on the basis of their need for palliative chemotherapy and did not include patients who were cured of their disease. Also, primary tumour TS expression may not give a good indication of the TS expression in metastatic lesions. The prognostic significance of TS protein expression in primary and metastatic lesions requires further evaluation.     

Direct inhibition of rat granulosa cell inhibin production by epidermal growth factor in vitro

The effect of epidermal growth factor (EGF) on inhibin production by rat granulosa cells has been investigated using a recently developed inhibin radioimmunoassay (RIA). Granulosa cells from intact immature diethylstilbestrol (DES)-treated rats were exposed to EGF (1-100 ng/ml) in the presence or absence of FSH for varying periods in vitro. An inhibitory effect of EGF on basal inhibin secretion was evident at day 2 of culture and was sustained over the subsequent 2 days. This action on basal inhibin secretion was dose-dependent, and maximal inhibition to 50% of control was observed at a dose of 100 ng EGF/ml at day 4. EGF also inhibited basal progesterone secretion in a similar manner. EGF caused a dose-dependent inhibition of FSH-stimulated inhibin secretion, with an ID50 (0.5 ng/ml, 0.08 nM) about one-eighth that in the absence of FSH. In addition, EGF also inhibited the stimulation of inhibin production by 8-Br-cAMP and prostaglandin E2. To exclude the possibility that EGF was toxic to the granulosa cells, several biochemical parameters related to cell growth were measured. EGF treatment did not alter cell number but slightly increased [3H]thymidine incorporation into cellular DNA. The effect of EGF on [35S]methionine incorporation into cellular protein was biphasic, being stimulatory at doses less than 10 ng/ml but inhibitory at 100 ng/ml. The present data have demonstrated a direct inhibitory effect of EGF on basal and FSH-stimulated inhibin production by granulosa cells suggesting an important regulatory role of this growth factor in the differentiation of ovarian function.     

Microcystic spinal cord degeneration causing posttraumatic myelopathy. Report of two cases

Two cases of progressive myelopathy occurring years after incomplete cervical spinal cord injury are presented. In both patients, the clinical features, as well as the "bull's-eye" appearance of the delayed computerized tomography (CT) myelography study and the circumscribed low density of the magnetic resonance image, were consistent with posttraumatic syringomyelia, but surgical exploration including intra-operative spinal sonography failed to reveal a syrinx. Although arachnoiditis was present in both patients, the striking abnormality found at surgery was the softened appearance and the microcystic degeneration of the cord. The microcystic spinal cord degeneration found in these cases represents a previously undescribed cause of late deterioration after spinal cord injury that may mimic the clinical, CT-myelographic, and magnetic resonance features of posttraumatic syringomyelia.     

Spontaneous activity of single neurones in the hypothalamus of rabbits during sleep and waking

1. A method is described for recording from single cells in the hypothalamus of unanaesthetized freely moving rabbits. Behaviour, bodily movement, skin and brain temperatures and e.e.g. were monitored.2. Patterns of unit firing during slow sleep, paradoxical sleep and waking were studied in several regions of the hypothalamus, thalamus and in the septum.3. Of the 144 cells analysed from waking to slow sleep, fifty-six (39%) decreased mean firing rates, thirty (21%) increased spike discharges and fifty-eight (40%) showed no marked change. Dorsal hypothalamic and massa intermedia thalamic cells fired in brief high frequency clusters during slow sleep with a characteristic ;bimodal' interspike interval histogram. Waking and paradoxical sleep abolished these cluster discharges with a concomitant change to an ;asymmetric' histogram.4. Of the thirty-two cells observed during the three states of waking, slow sleep and paradoxical sleep, a majority (twenty-five or 78%) showed their highest rates of spontaneous discharge during paradoxical sleep. Discharge rates of cells sometimes changed in the course of paradoxical sleep according to the presence or absence of phasic events such as myoclonic motor activity. Two hypothalmic cells were almost totally arrested during paradoxical sleep.5. Analysis of unit firing rates during spontaneous rises in brain temperature during waking and paradoxical sleep revealed that a majority of the neurones (22/24) changed their discharge rates in relation to behaviour rather than to brain temperature. Two cells did appear to respond specifically to the central thermal stimulus.6. Hypothalamic cells do not behave as a homogeneous population in relation to changes in the state of arousal of the rabbit. Spontaneous changes in cell discharge related to sleep-waking behaviour must be considered in any interpretation of hypothalamic unit activity as related to neuroendocrine or autonomic mechanisms.     

Hypothalamic temperature and the regulation of respiration of the ox exposed to severe heat

1. Thermodes have been chronically implanted in the hypothalamus of the ox.2. During exposure of the ox to severe heat stress, rapid shallow panting changed to slower deeper panting when hypothalamic temperature (T(hy)) was approximately 40.5 degrees C. With subsequent rapid ambient cooling, hypothalamic temperature declined rapidly, and there was a reversion to rapid shallow panting before respiration slowly returned to normal.3. When hypothalamic temperature and rectal temperature (T(R)) had reached about 40 degrees C, raising hypothalamic temperature to about 41.4 degrees C did not cause the onset of slower deeper panting.4. When hypothalamic temperature and rectal temperature had exceeded about 41 degrees C and slower deeper panting was well established, lowering hypothalamic temperature to about 40 degrees C did not cause any reversion to rapid shallow panting.5. When hypothalamic temperature and rectal temperature had reached about 40 degrees C, holding hypothalamic temperature at 40 degrees C did not prevent the onset of the slower deeper phase of panting.6. Under moderate heat stress when hypothalamic temperature was approximately 39.9 degrees C and rectal temperature was approximately 40.4 degrees C, raising hypothalamic temperature at the same rate as that which was associated with the onset of slower deeper panting during severe heat stress, resulted in a parallel increase in the respiratory frequency and fall in rectal temperature; there was no change to the slower deeper form of panting.7. Holding hypothalamic temperature at 41.7-42 degrees C during post-heat cooling did not prevent the reversion to rapid shallow panting.8. It was concluded that the change from rapid shallow to slower deeper panting during severe heat stress, and the reversion to rapid shallow panting during subsequent cooling, were not the result of changes in temperature of the hypothalamic thermoregulatory centres.     

A novel serine protease of the mammalian HtrA family is up-regulated in mouse uterus coinciding with placentation

This paper characterizes a novel gene, previously identified as uniquely regulated at implantation in mouse uterus. We cloned its full mRNA sequence encoding a serine protease possessing an IGF-binding domain and named it pregnancy-related serine protease (PRSP). PRSP is structurally similar to mammalian HtrA1 (56% amino acid similarity). Northern analysis revealed that the expression of PRSP mRNA was low before pregnancy, but it was increased at implantation and markedly up-regulated post-implantation. In-situ hybridization localized low levels of mRNA expression to the epithelium and stroma during very early pregnancy, but high expression to the decidual cells on day 8.5, primarily at the mesometrial pole where the placenta was forming. By day 10.5, PRSP mRNA was detected in the placenta. We also cloned an alternatively spliced PRSP mRNA that is expressed at a very low level. We located PRSP gene on chromosome 5 and established its intron/exon structure, which unambiguously explains how the two mRNA variants are produced through alternative splicing. Based on PRSP protein domain structure and its unique expression during pregnancy, we propose that PRSP plays an important role in the formation/function of the placenta.     

Inhibin and activin subunits are differentially expressed in endometrial cells and leukocytes during the menstrual cycle, in early pregnancy and in women using progestin-only contraception

Inhibins and activins are dimeric hormones which share common subunits and which have diverse endocrine and paracrine roles in regulating reproductive function. Endometrial expression of inhibin alpha, ssA and ssB subunits was examined by immunohistochemistry and in-situ hybridization, across the menstrual cycle and in early pregnancy. All three subunits were found to be expressed in endometrium, primarily by glandular epithelium in the early stages of the cycle. Following the onset of decidualization, expression of alpha, ssA and ssB subunits was up-regulated in decidualized stromal cells. A marked down-regulation of alpha subunit was detected in glandular epithelium, whilst expression of ssA and ssB subunits was maintained. This pattern was consistent in decidua from early pregnancy and additionally in endometrium from women using progestin-only contraceptives, either subdermal implants (Norplant((R))) or levonorgestrel-releasing intrauterine systems (Lng-IUS). Immunostaining was also observed for both ssA and ssB subunits in subpopulations of endometrial leukocytes, identified to be distinct subsets of macrophages, neutrophils and mast cells. Potential paracrine roles for activins may be envisaged in facilitating tissue remodelling during decidualization, in tissue repair following menstruation, and additionally in modulating premenstrual inflammatory events.