Ultrastructural immunolocalization of bone sialoprotein in guinea-pig osteoarthritis

OBJECTIVE: To compare the efficacy and tolerability of mibefradil and amlodipine in patients with uncomplicated mild-to-moderate essential hypertension. DESIGN: A double-blind, randomised, parallel group multicentre trial. METHODS: 239 patients received 50 mg mibefradil or 5 mg amlodipine for 4 weeks, followed by a forced titration to 100 mg mibefradil or 10 mg amlodipine for an additional 8 weeks. Patients then entered a 4-week withdrawal period either on therapy or switched to placebo. RESULTS: Statistically equivalent reductions in trough sitting diastolic blood pressure (SDBP) were observed after 12 weeks of once-daily treatment with 50/100 mg mibefradil (-11.5 +/- 8.2 mm Hg) and 5/10 mg amlodipine (-13.2 +/- 7.9 mm Hg). The number of patients with normalised SDBP (< or = 90 mm Hg) increased 23.3% in the mibefradil group and 19.5% in the amlodipine group (approximately 74% in both groups). Patients on mibefradil or amlodipine during the withdrawal period had significantly larger decreases in SDBP than those on placebo. Patients on mibefradil had a decrease in heart rate of 5.5 bpm. Patients on amlodipine had no change in heart rate; however, cessation of amlodipine was associated with a decrease in heart rate. CONCLUSIONS: Mibefradil was as effective as amlodipine in reducing BP; both compounds were effective treatments of hypertension.     

Expression of rat bone sialoprotein promoter in transgenic mice

Bone sialoprotein (BSP) is a major protein of the mineralized bone extracellular matrix that has been implicated in the nucleation of hydroxyapatite crystals. Our previous studies have demonstrated that BSP mRNA is expressed by differentiated osteoblasts, odontoblasts, and cementoblasts involved in de novo mineralized tissue formation in a tissue-specific and developmentally regulated manner. To determine the basis of the selective expression of the BSP gene, we have generated four transgenic mouse lines in which 2.7 kb of the rat BSP promoter ligated to a luciferase reporter gene has been stably integrated into the mouse genome. Assays of luciferase activities in 5-day-old animals has revealed consistently high levels in bone tissues with negligible activities in various other organs including kidney, liver, stomach, intestine, and spleen. In some animals, variable expression was observed in brain and skin. Temporal analyses revealed the highest luciferase expression in neonatal bones, with expression decreasing markedly with subsequent growth and development, as observed previously for the endogenous gene in rats. Immunohistochemical analysis of luciferase activity and in situ hybridization of luciferase mRNA in bone tissues show that differentiated osteoblasts express the highest levels of luciferase, consistent with the induction of endogenous gene expression. These studies demonstrate that the regulation of the BSP gene during osteoblastic differentiation, together with its tissue-specific, developmentally regulated expression, is primarily mediated within the 2.7 kb region of the promoter.     

Multiple subtypes of serotonin receptors are expressed in rat sensory neurons in culture

[3H]5-HT revealed the presence of serotonin receptors in cultured rat sensory neurons. [3H]5-CT binding was inhibited by cyanopindolol with an IC50 of 0.87 +/- 0.30 nM, suggesting the expression of the 5-HT1B receptor in these neurons. The presence of 5-HT1B receptors was confirmed by the displacement of [125I]Iodocyanopindolol binding by cyanopindolol with an IC50 of 2.43 +/- 0.81 nM. 5-HT1B receptors are the predominant type of serotonin receptors labeled by [3H]5-HT in cultured DRG neurons, representing approximately 60% of the specific [3H]5-HT binding sites. In addition, 5-HT1D and 5-HT2A receptor binding was also found in these neurons. RT-PCR analysis of RNA isolated from embryonic sensory neurons in culture confirmed the expression of 5-HT1B, 5-HT1D and 5-HT2A receptor mRNA. It also demonstrated the presence of 5-HT1F, 5-HT2C, 5-HT3, 5-HT4, 5-HT5A and 5-HT5B receptor mRNA and the absence of 5-HT1A, 5-HT1E, 5-HT2B, 5-HT6 and 5-HT7 mRNA. The identification of multiple subtypes of serotonin receptors expressed in cultured embryonic sensory neurons suggests that DRG neuronal cultures may be an excellent model to examine the direct effects of serotonin on the activity of these sensory neurons.     

Type I, II, and III inositol 1,4,5-trisphosphate receptors are unequally susceptible to down-regulation and are expressed in markedly different proportions in different cell types

The type I inositol 1,4,5-trisphosphate (InsP3) receptor can be rapidly depleted from cells during stimulation of phosphoinositide hydrolysis because its degradation is accelerated (Wojcikiewicz, R. J. H., Furuichi, T., Nakade, S., Mikoshiba, K., and Nahorski, S. R. (1994) J. Biol. Chem. 269, 7963-7969). The present study examines the regulatory properties of type II and III InsP3 receptors. Initially, the relative abundance of InsP3 receptors was defined in a range of cell types by quantitative immunoblotting. These studies showed that the proportions in which type I, II, and III InsP3 receptors are expressed differs greatly and that some cells (for example, AR4-2J rat pancreatoma cells) express all three receptors. Analysis of the effects of cholecystokinin and bombesin on AR4-2J cells showed that each of the InsP3 receptors could be down-regulated during activation of phosphoinositide hydrolysis, but that depletion of the type II receptor was limited. Such a discrepancy was also seen in rat cerebellar granule cells and was found to result from the type II receptor being relatively resistant to degradation. In conclusion, type I, II, and III receptors can all be down-regulated, but with different characteristics. As the relative abundance of InsP3 receptors is extremely variable, the extent to which activation of the down-regulatory process alters intracellular signaling will vary depending on which InsP3 receptors are expressed.     

Tumor labeling indices of primary breast cancers and their regional lymph node metastases

BACKGROUND: Tumor labeling index has emerged as a strong predictor of the clinical course of women with breast cancer. This study investigated whether labeling index of primary tumors correlates with labeling indices of concurrent regional node metastases. METHODS: With appropriate written consent, preoperative in vivo infusion of the thymidine analogue 5-bromodeoxyuridine (BrdUrd) was used to label 109 human breast cancers. Labeled S-phase cells were identified immunohistochemically with an antibody specific to DNA-incorporated BrdUrd. Labeling index was the fraction of labeled nuclei in 2000 tumor nuclei. For 30 women, there was sufficient cancer in axillary lymph nodes to compare labeling indices in primary breast cancer and regional lymph node metastases. RESULTS: The 30 women were from 25 to 82 years of age. Tumors were from 1 to 12 cm in size and there were from 1 to 26 positive nodes. Tumor labeling index ranged from 0.1% to 34%, (mean, 11.1%; median, 10.3%) and axillary lymph node metastasis labeling index ranged from 0.1% to 27.7% (mean, 10.8%; median, 10.0%). There was strong correlation between primary tumor labeling index and regional lymph node metastases labeling index (r = 0.82, with 95% confidence interval 0.65-0.91). The correlation persisted within subgroups according to age, tumor size, number of positive nodes, and hormone receptor status. Primary tumor and lymph node metastases labeling indices also had statistically similar relationships with age, level of hormone receptors, tumor size, and number of positive nodes. CONCLUSIONS: Primary tumor and regional node labeling indices correlate strongly; the relationship is not influenced by age, level of hormone receptors, tumor size, or number of positive nodes.     

Connexins are expressed in primary brain tumors and enhance the bystander effect in gene therapy

OBJECTIVE: Experimental brain tumor gene therapy with the herpes simplex virus thymidine kinase (HSV-tk) gene has demonstrated that not only HSV-tk transduced but surrounding non-HSV-tk transduced cells are killed when given ganciclovir. This so-called bystander effect has recently been shown to be dependent on connexin-mediated intercellular communication. To assess potential susceptibility to the bystander effect, we examined levels of connexin-26 and connexin-43 expression in a series of primary brain tumors. Connexin-26 expression has not previously been studied in primary brain tumors and connexin-43 expression has not been studied in nonastrocytic primary brain tumors. We also attempted to enhance the bystander effect in vitro by overexpressing connexin in tumor cells with high basal levels of connexin expression. METHODS: Western blot analysis and immunohistochemistry were used to determine levels of connexin-26 and connexin-43 expression in a series of primary brain tumors. Wild-type 9L gliosarcoma cells were transfected in vitro with the connexin-43 gene and the HSV-tk gene or the HSV-tk gene alone. The bystander effect of each transfectant was then assessed and compared. RESULTS: Most of the primary brain tumors tested, including low-grade astrocytomas, anaplastic astrocytomas, glioblastomas, oligodendrogliomas, gangliogliomas, meningiomas, and medulloblastomas, showed connexin-26 and connexin-43 expression. Bystander experiments revealed a significant enhancement of the bystander effect in the gliosarcoma cells transfected with connexin-43 and HSV-tk, as compared with gliosarcoma cells transfected with HSV-tk alone. CONCLUSION: Most primary brain tumors express connexin-26 and connexin-43. This suggests that most primary brain tumors may be susceptible to the bystander effect of HSV-tk gene therapy. The bystander effect can be enhanced in vitro by overexpression of connexin-43 in a cell line with a high basal level of connexin-43 expression.     

Estrogen receptors, progesterone receptors, and cell proliferation in human breast cancer

We grafted fetal thymi from wild-type mice into immunodeficient RAG-2-/- or class II-/-RAG-2-/- (class II MHC-) recipients and followed the fate of naive CD4+ T cells derived from the grafts. In both types of recipients, newly generated CD4+ T cells proliferated to the same extent in the periphery and rapidly filled the empty T cell compartment. However, CD4+ T cells in class II- recipients gradually decreased in number over 6 months. These results show that interactions between the TCR and class II molecules are not required for newly generated CD4+ T cells to survive and proliferate, but are necessary to maintain the size of the peripheral T cell pool for extended periods.     

Prognostic value of steroid receptors after long-term follow-up of 2257 operable breast cancers

The prognostic value of oestrogen receptor (ER) and progesterone receptor (PR) was estimated through a multicentric study of 2257 operable breast cancer patients followed up for a median of 8.5 years. None of the patients had received adjuvant therapy. The series included 33.3% stage I patients, 57.1% stage II, 5.7% stage IIIa and 2.4% stage IIIb. At the end point of the study 589 metastases and 537 deaths from cancer were recorded. Receptor measurements were performed by radiolgand assay according to a uniform protocol. A total of 68.8% of the tumous were ER positive and 54.0% PR positive ( > or = 10 fmol mg-1 cytosol protein). In univariate analysis, ER and PR status (positive/negative) were of prognostic value (P < 0.001) for the disease-free interval (DFI), the metastases-free interval (MFI) and the overall survival (OS). The OS of the patients after a first metastasis was also significantly different between ER-positive and -negative tumours (P < 0.001). In multivariate analysis (Cox proportional hazard model, 1665 patients), only the ER status showed a significant difference (P < 0.01) between positive and negative groups regarding the DFI, MFI and OS. By using Cox non-proportional, time-dependent models, we show that the predictive value of ER status of the primary tumour decreases by approximately 20% per year, losing its significance after 8 years of follow-up. Overall, when compared with TNM and histological grading, ER and PR status have a low prognostic value, their major interest remaining solely in the domain of therapeutic decision.     

Functional properties of glycine receptors expressed in primary cultures of mouse cerebellar granule cells

Expression of the glycine receptor was investigated in membranes prepared from primary cultures of mouse cerebellar granule cells and postnatal mouse cerebellum using the antagonist [3H]strychnine for ligand binding. Scatchard analysis of the binding data obtained from P17 cerebellum showed a single population of binding sites (K(D) approximately 6 nM) and [3H]strychnine binding to membranes prepared from cultured neurons and P17 cerebellum was found to have the same sensitivity to the glycinergic agonists glycine, beta-alanine and taurine. The development of [3H]strychnine binding sites in cultured cerebellar granule cells and cerebellum showed opposing profiles. [3H]strychnine binding to primary cultures increased significantly during the culture period whereas during development in vivo the number of binding sites decreased over time and was hardly detectable in the adult cerebellum. Release of preloaded D-[3H]aspartate evoked by 40 mM K+ from granule cells cultured for seven days was inhibited by glycine by about 50%. Beginning after seven days in culture the ability of glycine to inhibit transmitter release declined to no inhibition after 17 days in culture. Experiments with the non-competitive antagonist, picrotoxinin, showed no blocking effect of 150 microM picrotoxinin on the glycine-induced inhibition of transmitter release. This contrasted with the inhibitory effect of 100 microM picrotoxinin in whole-cell patch-clamp recordings on responses to 500 microM glycine (56% block). Furthermore, it was demonstrated that the amplitude of the glycine activated peak current had the same size after six to seven days and after 16-17 days in culture. Northern blot analysis, and co-injection of messenger RNA plus antisense oligonucleotides into Xenopus oocytes revealed glycine receptor alpha2 and beta messenger RNAs in the cultured granule cells. These findings suggest that granule cells in culture express glycine receptor isoforms containing alpha2 picrotoxinin-sensitive and alpha2/beta picrotoxinin-insensitive receptors.     

Osteosarcoma and other bone cancers

Streptokinase (SK), an extracellular protein from Streptococcus equisimilis, is secreted post-translationally by Escherichia coli using both its native and E. coli-derived transport signals. In this communication we report that cleavage specificity of signal peptidase I, and thus efficiency of secretion, varies in E. coli when SK export is directed by different transport signals. The native (+1) N-terminus of mature SK was retained when it was transported under the control of its own, PelB or LamB signal peptide. However, when translocation of SK was controlled by the OmpA or MalE signal peptide, Ala2 of mature SK was preferred as a cleavage site for the pre-SK processing. Our results indicate that compatibility of the leader peptide with the mature sequences of SK, which fulfills the requirement for a given secondary structure within the cleavage region, is essential for maintaining the correct processing of pre-SK. An OmpA-SK fusion, which results in the deletion of two N-terminal amino acid residues of mature SK, was further studied with respect to the recognition of alternative cleavage site in E. coli. The alanine at +2 in mature SK was changed to glycine or its relative position was changed to +3 by introducing a methionine residue at the +1 position. Both alterations resulted in the correct cleavage of pre-SK at the original OmpA fusion site. In contrast, introduction of an additional alanine at +4, creating three probable cleavage sites (Ala-x-Ala-x-Ala-x-Ala), resulted in the recognition of all three target sites for cleavage, with varying efficiency. The results indicate that the nature of the secondary structure generated at the cleavage junction of pre-SK, resulting from the fusion of different signal peptides, modulates the cleavage specificity of signal peptidase I during extracellular processing of SK. Based on these findings it is proposed that flexibility in the interaction of the active site of signal peptidase I with the cleavage sites of signal peptides may occur when it encounters two or more juxtaposed cleavage sites. Preference for one cleavage site over another, then, may depend on fulfillment of secondary structure requirements in the vicinity of the pre-protein cleavage junction.     

mu-Opioid and delta-opioid receptors are expressed in brainstem antinociceptive circuits: studies using immunocytochemistry and retrograde tract-tracing

Opioid-produced antinociception in mammals seems to be mediated in part by pathways originating in the periaqueductal gray (PAG) and the rostroventral medulla (RVM), and these pathways may include serotonergic neurons. In the present study, we examined the relationship of the cloned mu- and delta-receptors (MOR1 and DOR1, respectively) to PAG neurons projecting to the RVM, and RVM neurons projecting to the dorsal spinal cord. This was carried out by combining immunocytochemical staining for MOR1, DOR1, and serotonin with fluorescent retrograde tract-tracing. Of 133 retrogradely labeled cells in the RVM, 31% were immunoreactive for MOR1. Of the double-labeled cells, 41% also were immunoreactive for 5HT. Fifty-three percent of retrogradely labeled cells were apposed by DOR1-ir varicosities; 29% of the apposed cells were immunoreactive for 5HT. In the mesencephalon, cells retrogradely labeled from the RVM were usually surrounded by MOR1-ir structures; however, retrogradely labeled cells were never observed to be immunoreactive for MOR1. Similarly, retrogradely labeled cells in the caudal midbrain were seldom, if ever, labeled for DOR1; however, they frequently were apposed by DOR1-ir varicosities. Of 156 retrogradely labeled profiles from three rats, 52 (33%) were apposed by DOR1-ir varicosities. We conclude that both mu- and delta-opioid receptors could be involved in the antinociception mediated by the PAG-RVM-spinal cord circuit. In addition, opioids seem likely to have both direct and indirect effects on spinally projecting RVM cells in general, and on serotonergic RVM cells in particular.     

A comparison between radioligand and immunohistochemical assay of hormone receptors in primary breast cancer

The detection of oestrogen and progesterone receptor (ER and PgR) levels in human breast carcinoma has traditionally been performed using a biochemical radioligand binding method. This method has several disadvantages including the requirement for generous tissue samples, the production of radioactive waste products and the inability to exclude non-malignant cellular material from the assay process. An alternative method for detecting hormone receptors is available with the use of a monoclonal antibody specific for the ER or PgR receptor using immunocytochemical assay (ER-ICA or PgR-ICA). Although designed for use on frozen section material, with modifications this method can be used on paraffin sections of routinely fixed and processed tissue, on archival material and on very small specimens. Further, an objective assessment or scoring of staining intensity is possible using computerized video-image analysis. Forty-three cases of primary breast carcinoma, treated from 1989 to 1991 at Goulburn Valley Base Hospital, Shepparton were assessed for ER and PgR content using both the radioligand method and immunohistochemistry with video-image analysis, and the results were compared. Of the 43 cases, ER-ICA and ER had a concordance of 81% (P < 0.001, r = 0.58) and in 39 cases, PgR and PgR-ICA had a concordance of 87% (P < 0.001, r = 0.54). Because the sample for radioligand assay is of uncertain composition and the immunohistochemical stain can be scored specifically for malignant epithelium, a degree of discordance is thought to be mostly attributable to the limitations of the radioligand assay.     

Second primary cancers after cancers of the colon and rectum in New South Wales, Australia, 1972-1991

In this article, we present a survey on dental care and oral implantology in Beijing, China. The Chinese population comprises 1.2 billion or about 20% of the world's population. This survey shows: (i) there is a well-developed dental system in China, mostly operated by the Chinese government; (ii) in Beijing, there are 1328 dentists and oral surgeons and 515 special dental nurses working in dental departments of hospitals; (iii) about 2 million new patients visit the dentist every year; (iv) oral implantology is a new technology for the Chinese dentist and oral surgeon, as shown by the finding that in 1992, only 384 persons were treated with oral implants in a few hospitals in Beijing; however, most hospitals are interested in performing oral implantology in the near future; (v) imported implants are too expensive for Chinese patients, and therefore good qualified domestic implants and cheaper imported implants have a great market potential.     

The human homologue for the Caenorhabditis elegans cul-4 gene is amplified and overexpressed in primary breast cancers

Amplification is a key mechanism whereby a cancer cell increases the message level of genes that confer a selective advantage when they are overexpressed. In breast cancer, there are many chromosome regions present in multiple copies relative to overall DNA copy number (amplicons), and their target genes are unknown. Using differential display, we have cloned and sequenced the full coding region of a candidate amplicon target gene located on chromosome 13. This candidate is the human homologue of the Caenorhabditis elegans cul-4 gene, cul-4A, a member of the novel cullin gene family, which is involved in cell cycle control of C. elegans. cul-4A was amplified and overexpressed in 3 of 14 breast cancer cell lines analyzed, and it was overexpressed in 8 additional cell lines in which it was not amplified. The latter observation, indicating that its overexpression can occur by mechanisms other than gene amplification, suggests that cul-4A plays a key role in carcinogenesis. Moreover, cul-4A was found to be amplified in 17 of 105 (16%) cases of untreated primary breast cancers, and 14 of 30 cases analyzed (47%) were shown by RNA in situ hybridization to overexpress cul-4A. These results suggest that up-regulation of cul-4A may play an important role in tumor progression.     

Cyclic nucleotide phosphodiesterase 4 subtypes are differentially expressed by primary keratinocytes and human epidermoid cell lines

The monoclonal antibody 5B5 reacts with the beta subunit of proline-4-hydroxylase, the enzyme which catalyses the formation of 4-hydroxyl proline in collagen and other proteins with collagen-like amino acid sequences. This study aims to assess the production and tissue distribution of this enzyme in normal and diseased synovia from patients with various joint diseases, on the basis that it is a putative marker of collagen-producing cells and, therefore, in this context, of fibroblasts. Sections from five normal, 10 osteoarthritic (OA) and 26 rheumatoid arthritic (RA) synovia were labelled with a mouse monoclonal antibody to proline-4-hydroxylase. The enzyme was found to be expressed by a proportion of synovial intimal cells and by fibroblasts in the underlying connective tissue in normal, OA and RA synovia. Labelling was more pronounced in OA and RA cases. The intimal cells labelling positively showed type B synoviocyte morphology, which was confirmed by subsequent double immunolabelling with 5B5 and antibody against type IV collagen using immunocytochemistry and immunoelectron microscopy.     

Genetic epidemiology of breast and ovarian cancers

Most civilian and military air traffic control facilities in the United States use rapid rotation shift schedules. These schedules have generally been chosen for social reasons. Safety concerns have been raised because the air traffic controllers (ATCs) often carry an acute sleep debt onto the night-shift where they have little active work to do as they sit in the dark at the nadir of their circadian rhythms. This paper reviews advancing and delaying rapid shiftwork schedules, ATC workload factors as they relate to error rates and safety, and potential countermeasures. Recent studies indicate that ATC performance declines on the night-shift and that ATCs may be falling asleep while on-duty. There is indirect evidence that ATC error rates are highest on the night-shift. There are only limited studies which have evaluated potential countermeasures. The operational significance of the problems associated with ATC shiftwork is not yet clear. Further study is needed.     

N-methyl-D-aspartate receptors containing the NR2D subunit in the retina are selectively expressed in rod bipolar cells

N-methyl-D-aspartate receptor subunit messenger RNAs are widely expressed in the retina and several types of second and third order neurons are responsive to N-methyl-D-aspartate. Functional N-methyl-D-aspartate receptors are assembled from the NR1 subunit with at least one of the four NR2 subunit variants (NR2A-2D). We have analysed immunohistochemically the cellular distribution of N-methyl-D-aspartate receptors containing the NR2D subunit in the rat and rabbit retina. Using a subunit-specific NR2D antiserum, exclusively bipolar cells with somata localized close to the outer plexiform layer were labelled in both species. The axons were immunoreactive and arborized in the innermost inner plexiform layer. The morphology and localization of these cells, which were much more numerous in rat than in rabbit, suggested that they are rod bipolar cells. This was confirmed in both species by co-localization of the NR2D subunit immunoreactivity with protein kinase C-alpha, a selective marker for rod bipolar cells. At the subcellular level, a distinct polarization in the distribution of NR2D immunoreactivity was demonstrated by confocal laser scanning microscopy: staining was moderate in dendrites arborizing within the outer plexiform layer, intense at that pole of the soma facing the outer plexiform layer, and low in the portion of the soma embedded in the inner nuclear layer. Proximal axonal segments and axonal end-feet in the inner plexiform layer displayed the strongest NR2D subunit immunoreactivity. The axonal staining suggests that neurotransmission of the rod bipolar cells is modulated within the inner plexiform layer by N-methyl-D-aspartate receptors containing the NR2D subunit.