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Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of metabolic bone diseases. Although the molecular targets of bisphosphonates have not been identified, these compounds inhibit bone resorption by mechanisms that can lead to osteoclast apoptosis. Bisphosphonates also induce apoptosis in mouse J774 macrophages in vitro, probably by the same mechanisms that lead to osteoclast apoptosis. We have found that, in J774 macrophages, nitrogen-containing bisphosphonates (such as alendronate, ibandronate, and risedronate) inhibit post-translational modification (prenylation) of proteins, including the GTP-binding protein Ras, with farnesyl or geranylgeranyl isoprenoid groups. Clodronate did not inhibit protein prenylation. Mevastatin, an inhibitor of 3-hydroxy-3-methylglutatyl (HMG)-CoA reductase and hence the biosynthetic pathway required for the production of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, also caused apoptosis in J774 macrophages and murine osteoclasts in vitro. Furthermore, alendronate-induced apoptosis, like mevastatin-induced apoptosis, could be suppressed in J774 cells by the addition of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, while the effect of alendronate on osteoclast number and bone resorption in murine calvariae in vitro could be overcome by the addition of mevalonic acid. These observations suggest that nitrogen-containing bisphosphonate drugs cause apoptosis following inhibition of post-translational prenylation of proteins such as Ras. It is likely that these potent antiresorptive bisphosphonates also inhibit bone resorption by preventing protein prenylation in osteoclasts and that enzymes of the mevalonate pathway or prenyl protein transferases are the molecular targets of the nitrogen-containing bisphosphonates. Furthermore, the data support the view that clodronate acts by a different mechanism.

Map positions have been determined for 42 non-redundant Arabidopsis expressed sequence tags (ESTs) showing similarity to disease resistance genes (R-ESTs), and for three Pto-like sequences that were amplified with degenerate primers. Employing a PCR-based strategy, yeast artificial chromosome (YAC) clones containing the EST sequences were identified. Since many YACs have been mapped, the locations of the R-ESTs could be inferred from the map positions of the YACs. R-EST clones that exhibited ambiguous map positions were mapped as either cleavable amplifiable polymorphic sequence (CAPS) or restriction fragment length polymorphism (RFLP) markers using F8 (Ler x Col-0) recombinant inbred (RI) lines. In all cases but two, the R-ESTs and Pto-like sequences mapped to single, unique locations. One R-EST and one Pto-like sequence each mapped to two locations. Thus, a total of 47 loci were identified in this study. Several R-ESTs occur in clusters suggesting that they may have arisen via gene duplication events. Interestingly, several R-ESTs map to regions containing genetically defined disease resistance genes. Thus, this collection of mapped R-ESTs may expedite the isolation of disease resistance genes. As the cDNA sequencing projects have identified an estimated 63% of Arabidopsis genes, a very large number of R-ESTs (approximately 95), and by inference disease resistance genes of the leucine-rich repeat-class probably occur in the Arabidopsis genome.

The kinetics of inhibition of metastasis by the immunomodulator swainsonine (SW) is effective 1 to 3 days after administration. It is likely that SW's prolonged antimetastatic effect is due to its mitogenic property (spleenocytes isolated from animals treated with SW for 42-72 hours stimulated DNA synthesis that remained elevated for up to 3 days after removal of the drug from the drinking water). An analysis of SW in lymphoid (spleen and thymus) and highly perfused tissues was undertaken to determine if SW's sustained antimetastatic effect could be correlated to its retention. C57BL/6 mice received [3H]SW in drinking water for 24-72 hours and thereafter, received SW-free drinking for 24, 48, and 72 hours. Lymphoid and highly perfused tissues were analyzed for [3H]SW. At 24, 48, and 72 hours, spleen SW levels are, respectively, at least 2.33, 2.25, and 2.00 times greater than the perfused tissue; and thymus are, respectively, 1.44, 1.50, and 1.77 as great as the perfused tissue (kidney) with the highest SW level. These studies suggest that SW is predominantly retained for at least 72 hours, in lymphoid tissue. The targeting and retention of SW for lymphoid tissue days after removal of SW from animal drinking water is consistent with a) the immunomodulatory/mitogenic property and b) the sustained antimetastatic effect attributed to SW.

The potential of a new microanalytical method using magnetic beads (MBs) and commercial capillary electrophoresis (CE) instrumentation for performing enzymatic and inhibition assays, as well as for analysis of biological molecules such as antigens, substrates, etc., has been explored. A small quantity of magnetic beads containing immobilized biomolecules was injected into a neutral hydrophilic-coated fused-silica capillary. The short plug (2-3 mm) of beads was held fixed by a magnet placed in the cartridge of the CE system, without the use of frits. The beads could be replaced after each run, eliminating the need to regenerate the solid support. Two protocols were used for analysis: sequential injection (SI) and SI followed by isotachophoretic (ITP) focusing. Alkaline phosphatase (AP) and HIV-protease were used to demonstrate the SI procedure for enzymatic and inhibition assays. The second protocol, SI/ITP, was employed to quantitate an antigen (mouse mAB) using antibodies (sheep IgG towards mouse AB) immobilized on the beads. The MB-CE method, requiring only femtomole (fmol) quantities of material, can potentially be employed in diagnostic and forensic assays, kinetic studies and searching for inhibitors, ligands, receptors, etc.

Within the pH range 2-12, the monoclonal chimeric antibody BR96 can be separated into one to five isoforms by micellar electrokinetic capillary chromatography (MECC). The distribution of the immunoglobulin between these isoforms is pH dependent and apparently reversible. Some of the changes in the electrophoretic profile are represented by alterations in the immunoglobulin secondary structure. MECC and CD data demonstrate that, in other cases, differences in electrophoretic mobilities of the intact and acid-stressed antibody molecules were not due to differences in the ionization of the protein functional groups or changes in secondary structure, but rather resulted from differences in the exposure of the molecule's structural elements to the solvent. The results indicate that the interaction of the isoforms with sodium dodecyl sulfate micelles plays a crucial role in MECC isoform separations. The formation of analyte-micelle complexes was postulated to make electrophoretic mobilities, especially of large protein molecules, susceptible to subtle conformational changes that are not detectable by other methods.

Assays for detecting and measuring antinuclear antibodies (ANA) in dogs were compared. They included the indirect immunofluorescence test, using rat liver as substrate, and ELISAs for three nuclear antigens: double-stranded DNA, single-stranded DNA, and histone. There was no correlation between the ANA titre and antibodies to the three nuclear antigens. Analysis of ANA in different arthropathies showed no specific disease association. HEP-2 cells showed no fluorescence reaction with either ANA-positive or ANA-negative dog sera. Western blotting produced too complex a pattern to identify specific antigens. The antigens that reacted with ANA in dogs were not identified; there is either a broad range of reactivities or non-specific binding of immunoglobulins.

A procedure is presented for the separation of underivatized oligosaccharides by capillary electrophoresis (CE) with a phytic acid-borate buffer system. The presence of the phytic acid ion-pairing agent greatly increases resolution between oligosaccharides in the complex mixtures studied, which was demonstrated by the separation of oligosaccharides originating from various immunoglobulin G antibodies and CTLA4Ig, a biologic fusion protein. The conditions also resolve neutral oligosaccharides, usually a major CE limitation. High-performance anion-exchange chromatography with pulsed amperometric detection, a standard technique for oligosaccharide and sugar analysis, is used as a reference method to analyze some of the complex oligosaccharide mixtures.

Postmenopausal osteoporosis, the most common bone disease in the developed world, is associated with estrogen deficiency. This deficiency induces increased generation and activity of osteoclasts, which perforate bone trabeculae, thus reducing their strength and increasing fracture risk. Estrogen replacement prevents these effects, indicating that estrogen negatively regulates osteoclast formation and function, but how it does this is unclear. Because functional osteoclast life span and thus the amount of bone that osteoclasts resorb could also be enhanced following estrogen deficiency, and since sex steroids regulate apoptosis in other target tissues, we investigated whether estrogen may affect osteoclast function by promoting apoptosis. 17 beta-Estradiol promoted apoptosis of murine osteoclasts in vitro and in vivo by two- to threefold. Tamoxifen, which has estrogenic effects on bone resorption, and transforming growth factor-beta 1 (TGF-beta), whose production by osteoblasts is increased by estrogen, had similar effects in vitro. Anti-TGF-beta antibody inhibited TGF-beta-, estrogen- and tamoxifen-induced osteoclast apoptosis, indicating that TGF-beta might mediate this effect. These findings suggest that estrogen may prevent excessive bone loss before and after the menopause by limiting osteoclast life span through promotion of apoptosis. The development of analogues to promote this mechanism specifically could be a useful and novel therapeutic approach to prevent postmenopausal osteoporosis.

Interactions between articular chondrocytes and components of the extracellular matrix are of potential importance in the normal function of cartilage and in the pathophysiology of arthritis. Little is known of the basis of these interactions, but cell adhesive molecules such as CD44 are likely to be involved. Immunohistology using six well-characterized anti-CD44 monoclonal antibodies demonstrated standard CD44 isoform (CD44H) expression by all chondrocytes in normal and osteoarthrotic (OA) cartilage but absence of the CD44E variant. Polymerase chain reaction (PCR) of reverse transcribed mRNA from monolayer cultures of normal and OA chondrocytes using primer sequences which span the region containing variably spliced exons produced a predominant band representing the standard form of CD44, which lacks the variable exons 6-15 (v1-v10). No product was seen at the expected size of the epithelial variant of CD44 (CD44v8-10). Use of exon-specific primers, however, showed expression of variant exons resulting in multiple minor isoforms. Standard CD44 was also shown to be the predominantly expressed isoform identified by immunoprecipitation, but human articular chondrocytes did not adhere to hyaluronan in vitro. Chondrocyte CD44 may function as an adhesion receptor for other matrix molecules such as fibronectin or collagen.

Methods have been developed for the CE and HPLC analysis of CTLA4Ig, an immunoglobulin fusion protein. Two different LC approaches, size-exclusion (SEC) and "mixed-mode" ion-exchange (ABx), were developed along with a CE method that uses a micellar electrokinetic chromatographic buffer consisting of borate ions, sodium dodecyl sulfate and acetonitrile. These assays measure the presence of several CTLA4Ig-related species, and the observed changes resulting from multiple modes of degradation. In an attempt to identify possible degradation products, collections were taken from the ABx and SEC liquid chromatographic systems and further analyzed by matrix-assisted laser desorption time-of-flight (MALDI TOF) mass spectrometry. Multiple species were detected covering a wide molecular mass range. In addition, the CE method was used to study conformational kinetics between two forms of CTLA4Ig and to estimate the activation energy of the conformer-conformer transition. Pseudo-first-order reaction kinetics were demonstrated for CTLA4Ig samples stressed with papain, H2O2, and sodium dodecyl sulfate/heat.

Monitoring the stability of immunoglobulin G (IgG) type antibodies is a crucial analytical issue spanning a wide variety of immunological/biotechnological studies, which includes the analysis of conjugated IgG's for drug delivery. Capillary electrophoresis (CE) has proven valuable for the analysis of proteins and has the potential to separate and detect native antibody components. An ideal complement to CE, which is capable of providing the desired detection specificity to provide species identification information, is matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Utilizing these two techniques we have developed an antibody examination procedure and monitored the degradation of an internalizing chimeric (human/mouse) monoclonal antibody (BR96). Electropherograms of the antibody after up to 166 h of thermal stress are presented; MALDI mass spectra of the stressed antibody were acquired at the same time points. At 166 h, the percentage of ionization carried by the intact antibody molecular ions M+, M2+, etc., had clearly decreased, while that due to additional ion species had significantly increased. Ions corresponding in mass to loss of one light chain, loss of an Fab arm to yield an Fab/c type fragment, and formation of separated heavy-chain and light-chain moieties were observed. Several of these fragments result from simple disulfide linkage disruption. In addition, species less in mass than common antibody subunits were also observed, demonstrating peptide as well as disulfide bond cleavage. The observation that a small number of well-defined species were formed during the study suggests that the cleavage induced by thermal stress is very site-specific within the IgG.

Bisphosphonates inhibit bone resorption and are therapeutically effective in diseases of increased bone turnover, such as Paget's disease and hypercalcemia of malignancy. The mechanisms by which they act remain unclear. Proposed mechanisms include inhibition of osteoclast formation from precursors and inhibitory or toxic effect on mature osteoclasts. We have developed a new in vitro model to study osteoclast survival and in this paper present in vitro and in vivo evidence that may explain both the observed reduction in osteoclast numbers and in bone resorption by mature osteoclasts, namely that bisphosphonates induce programmed cell death (apoptosis). Three bisphosphonates (risedronate, pamidronate, and clodronate) caused a 4- to 24-fold increase in the proportion of osteoclasts showing the characteristic morphology of apoptosis in vitro. This observation was confirmed in vivo in normal mice, in mice with increased bone resorption, and in nude mice with osteolytic cancer metastases, with similar-fold increases to those observed in vitro. Of the three compounds, risedronate, the most potent inhibitor of bone resorption in vivo, was the strongest inducer of osteoclast apoptosis in vitro. Osteoclast apoptosis may therefore be a major mechanism whereby bisphosphonates reduce osteoclast numbers and activity, and induction of apoptosis could be a therapeutic goal for new antiosteoclast drugs.

Four major isoforms of the BR96 antibody were separated by micellar electrokinetic capillary chromatography. Heat-induced reversible isoform interconversions were observed at 70 degrees C, and after extended incubation at 80 degrees C, all species irreversibly transformed into a new single peak. In the presence of sodium dodecyl sulfate (1.0 mg/mL), the isoform transformations occurred at lower temperatures without altering the separation pattern. Size exclusion chromatography analysis detected no aggregation at temperatures below 80 degrees C. Parallel circular dichroism measurements indicated significant conformational changes at 70-80 degrees C. The parallelism between isoform transformations and secondary structure changes allows consideration of CE-separated isoforms of BR96 antibody as conformers, an equilibrium between which can be shifted by different physicochemical factors such as elevated temperatures and amphiphilic surfactants.

Aims: To determine the histological features in CIN3 associated with or predictive of subsequent microinvasion., Methods: The histological appearances of CIN3 accompanying 120 cases of microinvasive carcinoma of the uterine cervix were retrospectively studied. Major features were defined as those present in greater than 80% of cases of microinvasive carcinoma (MICA) and less than 10% of control cases of CIN3. One hundred cases of CIN3, 36 showing all, and 64 lacking all, of the major features associated with microinvasion, as defined in the retrospective study, were prospectively studied. Deeper levels were cut to exclude the presence of microinvasion in the original biopsy specimen and negative cases were followed up for a period of up to 18 months in order to assess rates of recurrence or progression., Results: The major features identified in CIN3 associated with microinvasive carcinoma were extensive involvement of surface epithelium and deep endocervical crypts by expansile CIN3, luminal necrosis, and intraepithelial squamous maturation. Other features more commonly present in MICA associated CIN3 than in controls included frequent mitosis and apoptosis, pericryptal concentric fibroplasia, pericryptal inflammatory infiltrate, pronounced cellular pleomorphism, nuclear changes (distinct nucleoli and chromatin clearing), and the emergence of streams of darkly stained spindle cells oriented at right angles to the basement membrane. In the prospective study 83% of cases illustrating the major MICA associated features revealed evidence of MICA or frank invasion either on serial sections of the original biopsy or on subsequent biopsy. None of the 64 cases of CIN3 that lacked these features showed evidence of invasion on serial sections or on further follow up over 18 months., Conclusions: The data strongly suggest that cases of CIN3 which have a higher probability of association with or rapid progression to invasive disease can be identified. When these features are present in a biopsy specimen of CIN3, serial sections should be performed to exclude the presence of microinvasion. Closer clinical follow up of these patients may be needed.

A procedure for the analysis of mixtures of underivatized, cleaved O-linked and N-linked oligosaccharides by capillary electrophoresis has been developed. The species of interest are separated by two borate-based buffer systems with an uncoated capillary. The procedure is applied to hydrazinolysis-released oligosaccharides from mouse, rat, sheep and human IgG protein samples which are examined within 6 minutes. Selectivity with respect to sample variation is demonstrated by analysis of thermally stressed samples.

A 13-year-old boy presented with right-sided gynecomastia. Histological examination of the excised specimen revealed distention of ducts by papillary configurations of proliferating epithelium. The boy had no known family history of breast disease and physical examination revealed minor dysmorphic features, but a karyotype was normal. The possible relationship of this lesion to other proliferative lesions described in the pubertal male breast is discussed.

Epithelial cell-basement membrane interactions are important in maintaining tissue architecture and function, and the anatomical and functional relationships between epithelial cells and their basement membranes are clearly altered in malignancy. These interactions are thought to be largely mediated by the integrins, a family of heterodimeric transmembrane glycoproteins, each consisting of an alpha and a beta chain. Epithelial integrins mainly belong to the beta 1 (VLA) subfamily, which forms receptors for matrix macromolecules such as fibronectin, laminin, and collagen. There is evidence that integrin expression changes in some epithelial malignancies, possibly in relation to invasive potential. Integrin expression in cervical neoplasia was studied by immunohistochemical examination of prospectively collected colposcopic biopsies. Well-characterized monoclonal antibodies against beta 1-4, alpha 1-6, and alpha V integrins were used to examine normal, koilocytic, and dysplastic cervical squamous epithelium, and invasive squamous carcinoma. beta 1, beta 4, alpha 2, alpha 3, alpha 6, and alpha V were expressed by the basal layer of normal cervical squamous epithelium and by dysplastic cells in CIN (cervical intraepithelial neoplasia) 1 and 2, with none being lost and no new chains acquired. In CIN3, these integrins were either expressed throughout the ectocervical epithelium or restricted to the basal layer. In the latter cases, integrin expression was retained to a greater degree by dysplastic squamous epithelium within endocervical glands. These patterns could not be correlated with age or smear history in the cases examined. Patterns of integrin expression in neoplastic cervical epithelium therefore differ from those of normal cervical epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Vitreoscilla hemoglobin (VHb) has been expressed in Saccharomyces cerevisiae, and its influence on yeast aerobic metabolism has been investigated. New expression vectors were constructed to express VHb constitutively under the control of the ADH-1 promoter. The presence of VHb was shown by Western blot analysis. VHb has been shown to localize predominantly in the cytoplasm. Batch fermentation results indicated that the wild-type strain expressing VHb exhibited a shift in the carbon flux toward ethanol production, with no significant alteration in the specific growth rate. This effect was not observed if cells were grown under respiration inhibition, indicating that the metabolic effect of VHb is likely linked to respiration. Expression of VHb in the adh degrees strain MC65-2A, which produces ethanol only via a respiration-coupled pathway, revealed that ethanol production was decreased and cells reached a higher final cell density in a culture of the VHb-expressing strain. Growth enhancement due to expression of VHb was observed only during the final stage of culture growth when the acetaldehyde produced during the first growth phase was used as a substrate. This metabolic effect of intracellular VHb was seen more clearly in an acetaldehyde fed-batch fermentation in which VHb-expressing cells grew to at least 3-fold higher final cell density. These results suggest that the action of VHb is likely linked to electron transfer.

CD44 is a transmembrane glycoprotein with cell-cell and cell-matrix adhesion functions that is expressed by a wide variety of cell types and has a number of known biologic functions. Because of its ability to bind matrix macromolecules, such as fibronectin, collagen, and hyaluronate, we investigated the possibility that it is expressed by the cells of bone, the matrix receptors of which are largely unknown. Immunohistochemical study of a variety of sources of human bone was carried out using a panel of six well-characterized anti-CD44 monoclonal antibodies. Osteocytes strongly expressed CD44, whereas osteoblasts and lining cells were negative. Osteoclasts and periosteal cells also expressed CD44, although not as strongly as osteocytes. These patterns of staining were observed with all six antibodies. These results demonstrate that acquisition of CD44 immunoreactivity is a sensitive marker of osteocytic differentiation and raise the possibility that CD44 acts as a cell matrix receptor in bone.

Hyaline eosinophilic globules have so far been described in a restricted variety of tumour types. We have noted their presence in a variety of gynaecological malignancies, in particular mixed Müllerian tumours and other epithelial ovarian tumours. We therefore studied their incidence and distribution in a series of malignant, borderline and benign epithelial ovarian tumours, and endometrial and endocervical adenocarcinomas. Hyaline eosinophilic globules were found in all 30 mixed Müllerian tumours from various sites in the female genital tract, 22 of 30 clear cell carcinomas, seven of 30 serous, two of 30 mucinous, and one of 30 endometrioid carcinomas examined, and were also seen in metastases from these tumours. They were present in only two of 25 borderline serous, one of 25 borderline mucinous tumours, and in four of 50 benign serous and one of 50 benign mucinous tumours. The globules were not found in any of 25 Brenner tumours examined, nor in 30 endometrial or 30 endocervical adenocarcinomas. The globules were periodic acid-Schiff positive after diastase, stained positively with PTAH, and were immunoreactive for alpha-1-antitrypsin. This study therefore demonstrates that eosinophilic globules are not specific for any particular tumour. However, their frequency in malignant mixed Müllerian tumours suggests that this diagnosis should be carefully excluded whenever these globules are present in epithelial tumours of the female genital tract.

A DNA vector for expressing an oxygen-binding heme protein (Vitreoscilla hemoglobin, or VHb) in filamentous fungi was constructed and introduced into a cephalosporin C-producing strain of Acremonium chrysogenum. Expression of VHb in transformants was demonstrated by Western immunoblot analysis and by increased carbon monoxide binding activity of cell extracts. Several VHb-expressing transformants produced significantly higher yields of cephalosporin C than control strains in batch culture experiments. Using the same vector system, VHb was also expressed in the related fungus Penicillium chrysogenum.

Integrins are a family of heterodimeric transmembrane glycoproteins that are known to mediate cell-cell and cell-matrix interactions. Members of the VLA (very late activation) family, which consists of beta 1 integrin in association with the VLA alpha chains (alpha 1-6), mediate adhesion of a wide range of cells to matrix proteins, such as fibronectin, collagen, and laminin, and may therefore be important for cell-matrix interactions in bone. Integrin expression in human bone was studied immunohistochemically using cryostat sections of fracture callus, tumor-associated reactive bone, and neonatal costochondral junctions, with a panel of well-characterized antibodies against beta 1-4 integrins, alpha 1-6 and alpha V integrins, and the alpha V beta 3 dimer (the classic vitronectin receptor). All cell types present in bone expressed beta 1 and alpha 5 integrins; a subpopulation of osteoblastic cells expressed alpha 4. The alpha V was uniformly expressed by osteoblasts but was heterogeneously expressed by osteocytes. Osteoclasts also expressed alpha 2, alpha V, and alpha V beta 3. These results demonstrate differential expression of a restricted range of integrins in bone. This supports the possibility that integrins may mediate the differing interactions of cells of the osteoblast and osteoclast lineages with the matrix of bone.

Serological surveys of common and grey seals were performed to monitor the spread of the phocid distemper outbreak in 1988 and its effects on the host immune response. The disease affected mainly common seals; grey seals were largely unaffected but did have anti-CDV antibodies which developed during 1988. Canadian seals, both common and grey, showed evidence of a morbillivirus infection but without the apparent mortality observed in British common seals. Common seal circulating immunoglobulin levels were not greatly affected during the outbreak, although there was some reduction in the most severely affected pups. Vaccination of common seals with CDV antigens elicited high titres of antibodies and produced clinical protection. Vertical transmission of host immunity was shown in grey seals although the longevity of protection has not been determined. The large population of common seals which are not immune emphasises that such an epizootic could have serious consequences again in the future.

Expression of integrins, a family of cell adhesion proteins, by human articular cartilage chondrocytes in vivo has been studied by immunohistological techniques using cryostat sections and a panel of anti-integrin monoclonal antibodies. Chondrocytes strongly expressed beta 1 and alpha 5 integrin subunits, known to form the classical fibronectin receptor, VLA-5, strongly. alpha 1 was expressed more weakly and variably; alpha 3 was expressed by occasional cells only. Chondrocytes did not express beta 2, beta 3 or other integrin alpha subunits tested.

Lymphocyte populations in the synovial membranes of dogs with canine rheumatoid arthritis (CRA) were investigated by immunohistochemical staining techniques. T-lymphocytes were the predominant cell type distributed throughout the supporting layer of the synovial membranes. B-lymphocytes expressing IgG were seen far more commonly than those expressing either IgA or IgM. Synovial membrane biopsies from normal and osteoarthritic joints did not have the marked cellular infiltrates seen in joints with CRA. The synovial immunohistopathological features in dogs with CRA are similar to those seen in human rheumatoid arthritis.

We report a case of sacrococcygeal chordoma initially diagnosed by fine-needle aspiration cytology. The value of awareness by cytopathologists of the histology of soft tissue tumours is emphasized.

Interleukin 6 (IL-6) probably plays a central role in the acute phase response and in haemopoiesis and may be involved in the control of bone turnover. We have studied the release of IL-6 from human trabecular bone cells treated with a variety of stimuli using a specific bioassay. In serum free medium, unstimulated human osteoblast-like cells produced IL-6 in the range of 1000-2050 pg/ml/24 h. Recombinant human interleukin 1 (IL-1 alpha) (10(-13)-10(-11) M), tumor necrosis factor alpha (TNF alpha) (10(-9)-10(-7) M) and lipopolysaccharide (5-500 ng/ml) all stimulated release of IL-6 from human bone cells. Maximal levels of 17,000 pg/ml were observed using the highest concentration of IL-1. 1,25(OH)2D3 and PTH did not stimulate IL-6 release. Using a specific sheep antihuman IL-6 antibody, all IL-6 activity could be neutralized. In parallel studies, ROS 17/2.8 rat osteosarcoma cells released around 50 pg/ml of IL-6 under basal conditions which were increased to a maximum of 900 pg/ml by treatment with PTH (10(-9) M). The cytokines were less effective and 1,25(OH)2D3 again had no effect. Modulation of expression of IL-6 mRNA in human osteoblast cells was examined using a human complementary deoxyribonucleic acid probe. The mRNA was constitutively expressed, and IL-1 (10(-11) M) and TNF (10(-7) M) induced further mRNA expression within 2 h, which was sustained over 24 h. 1,25(OH)2D3 (10(-7) M), IL-6 (2000 pg/ml), and PTH (10(-9) M) exerted no effects at any time point. Dexamethasone (10(-6) M) suppressed both basal and IL-1- and TNF-induced IL-6 mRNA expression. IL-6 receptor mRNA was constitutively expressed but was not regulated by any of the above agents. It is clear that rodent and human osteoblasts differ in their production of IL-6 and its modulation. These data support the hypothesis that IL-6 is produced locally in human bone by osteoblasts under the direction of other cytokines. This could have implications in bone remodeling, haemopoiesis, and systemic responses to local injury.

Secondary metabolite production by Streptomyces is often highly sensitive to oxygen supply, which can be limiting in large-scale fermentations. In an attempt to improve oxygen utilization by the cells, we expressed a heterologous bacterial hemoglobin gene in Streptomyces coelicolor and Streptomyces lividans. Hemoglobin expression was demonstrated by immunoblot analysis and carbon monoxide binding activity. In batch fermentations run under reduced aeration, the expression of hemoglobin in S. coelicolor resulted in a ten-fold increase in specific yields of the aromatic polyketide, actinorhodin. Actinorhodin yields were also much less sensitive to aeration conditions in the hemoglobin-expressing strain. In addition, hemoglobin-expressing S. lividans cells grown under reduced aeration had higher final cell densities and exhibited greater oxygen consumption rates than non-expressing cells.

Present understanding of the cellular mechanism of action of bisphosphonates as in vitro and in vivo inhibitors of bone resorption is reviewed. This mechanism appears to involve the osteoclast at more than one stage of its maturation process, and way well be receptor-mediated, although the molecular mechanism itself is thus far unknown.

Recombinant human interleukin-6 (IL-6) was assessed for its ability to stimulate bone resorption in prelabeled mouse calvariae in vitro. IL-6 had no effect on bone resorption at concentrations ranging from 300 to 10,000 U/ml (3-1000 pg/ml). Neither the presence of indomethacin nor prolonged incubation periods (96 h) affected this result. IL-6 did not affect resorption stimulated by human recombinant IL-1 alpha (rIL-1 alpha) but inhibited resorption stimulated by parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. rIL-1 alpha, PTH, and 1,25-(OH)2D3 induced IL-6 release by calvariae. We conclude from these studies that IL-6 does not stimulate bone resorption in neonatal mouse calvariae. However, it may act as a locally produced inhibitor and therefore a paracrine regulator of bone resorption induced by osteotropic hormones. IL-6 could also function as a long-range stimulator of systemic reactions and acute-phase responses to local inflammatory and neoplastic lesions in bone.

Activated immune cells release cytokines which modulate the activity of bone cells in vitro. Expression of major histocompatibility complex (HLA in humans) class II determinants on bone surface cells may be important in local immune cell activation. In this study, expression of HLA-DR and DQ by cultured human bone cells (HBC) derived from normal trabecular bone surfaces was assessed by fluorescence-activated cell sorter (FACS) analysis and immunoperoxidase techniques using monoclonal antibodies. A subset of HBC (10-30%) expressed DR constitutively while 5-15% displayed DQ during long-term culture. HBC lacked a number of monocyte and lymphocyte markers. In addition, both DR+ and DR- HBC (FACS separated) produced osteocalcin stimulated by 1,25-dihydroxyvitamin D2 (1,25(OH)2D3). This suggests that both phenotypes belong to the osteoblast lineage. The number of DR+ HBC was increased by interferon-gamma (IFN gamma; 40-95% DR+ cells) whereas DQ+ HBC remained unchanged or was slightly increased (5-20% DQ+ cells). Moreover, 1,25(OH)2D3 enhanced IFN gamma-induced DR expression and at high concentration (10(-7) M) augmented DR expression by itself. Other major osteotropic factors, parathyroid hormone, interleukin 1, and calcitonin, did not affect HBC DR expression. The findings suggest that HBC may participate in activation of the immune system and that some osteotropic factors may regulate this function.

Immunoglobulin concentrations were measured in the serum and colostrum of adult and pup Grey seals from North Rona in the outer Hebrides and the Isle of May in the Firth of Forth. IgG was shown to be the main immunoglobulin and two subclasses were identified. Serum immunoglobulin content was low in the week-old pups and increased up to 5 weeks, although it remained substantially lower than in the adults. Colostral immunoglobulins were high and cannot explain the low values in the pup serum. It is considered that the increased opportunistic infections seen in Grey seal pups may be related to this low immunoglobulin status.

A total of 450 yeast isolates were obtained from up to 34 sites on 59 human subjects. The yeasts were characterized using morphological features and assimilation tests. Ten species were identified but, of these,Candida albicans andCandida parapsilosis were the most common and accounted for 84% of the isolates. An examination of the biotypes of the various species indicated a much greater diversity in the yeast microflora than that detected by species identifications alone. Fifty-five biotypes were differentiated and it is suggested that these could be regarded as distinct taxonomic or ecological entities.

The titrimetric determination of ascorbic acid in the presence of a variety of potentially physically and chemically interfering species in commercial liquid diets is presented. The titrant and indicator was a solution of 2,6-dichlorophenol indophenol. Iron(II), copper(II), cysteine, glutathione, sulfite, and tin(II) do not interfere.

A repeated sequence from the Crithidia fasciculata nuclear genome has been isolated which is homologous to the mini-exon genes of other kinetoplastid protozoa. Sequence analysis of the 417 bp monomeric unit confirmed the presence of a 35 nt sequence within the repeat that is 77% homologous with the Trypanosoma brucei 35-mer mini-exon or spliced leader sequence. The repeat is present at approximately 250 copies per cell and is organized into one, or a few, large head to tail tandem clusters predominantly on a single chromosome. The mini-exon repeat unit hybridizes to a major 84 nt and a minor 87 nt poly (A)- steady state transcript, the first 35 nts of which comprise the mini-exon sequence found at the 5' end of mRNAs in several other kinetoplastid species. The 3'-termini of the transcripts map to positions on the DNA sense strand directly preceeding a stretch of 8 thymidine residues. Crithidia represents the most primitive kinetoplastid species which apparently possesses a discontinuous type of mRNA processing, implying that this represents a conserved feature in possibly all genera of kinetoplastid protozoa.

A vaccination study was conducted for infectious bovine keratoconjunctivitis (IBK) in 440 purebred Hereford cattle (cows and their newborn calves) of the USDA Meat Animal Research Center cattle herd at Clay Center, Ne. The cattle were allotted to 4 groups: 60 calves were vaccinated with an autogenous Moraxella bovis bacterin (group 1); 60 calves that were matched with group 1 calves were designated nonvaccinated matched controls (group 2); 99 calves were peer group nonvaccinated controls (group 3); and 219 cows, the dams of the calves, were nonvaccinated consorts (group 4). The infection rates in cattle groups 1, 2, 3, and 4 during the summer were 96.6, 98.3, 100, and 79.1%, respectively, and the disease rates were 90, 93, 85, and 20%. The infection and the disease rates were significantly (P less than 0.01) different between claves and cows. The disease rate was also significantly different between older and younger cows. A larger percentage of the affected calves and cows had mild or moderate (61%) signs of IBK rather than severe (39%) signs. The rate of body weight gain was reduced in calves with severe signs of IBK. The results seemed to indicate that little would be gained by vaccinating cattle against IBK under the conditions of study.

Several properties of a new oxygen-regulated promoter, OXYPRO, were tested in small-scale Escherichia coli cultures. Using OXYPRO, maximal activity of a reporter gene encoding chloramphenicol acetyltransferase (CAT) occurred in cultures that were tightly capped immediately after inoculation. This is probably a result of the reduced oxygen concentration attained in capped cultures, a condition known to be required for OXYPRO induction. CAT levels were significantly higher when the cells were grown in a glycerol-based medium. Similar levels of CAT expression were obtained when OXYPRO was compared to the trp-lac (tac) promoter. In addition, regulated expression of CAT occurred in a wild type strain of E. coli, suggesting that OXYPRO will be useful in most E. coli strains. Thus, OXYPRO provides a simple, inexpensive, and unobtrusive method to achieve high levels of cloned protein expression in most strains of E. coli. OXYPRO is available in a high copy plasmid with a convenient multiple cloning site for the insertion of genes for direct expression in E. coli.

Mycobacterium paratuberculosis, the cause of Johne's disease, was isolated from the feces of a donor bull in an artificial insemination stud. During isolation and observation for 21 months, the organism was recovered from all of 26 fecal samples and from 8 of 31 semen samples. At necropsy, it was isolated from the intestine and adjacent lymph nodes, lung, spleen, seminal vesicles, and prostate gland but not from the testicles. We concluded that routine fecal cultures at bull studs will reveal infected bulls before they become genitally infected and shed M paratuberculosis in semen.

Mutants of Crithidia fasciculata, resistant to Actinomycin D, Tubercidin, Crystal Violet, 6-Azauridine, and 5-Fluorouracil were obtained and characterized. The mutants were stable when maintained under nonselective conditions, and could be grouped in several subclasses on the basis of cross-resistance to other drugs. Actinomycin D- and Crystal Violet-resistant mutants appeared spontaneously whereas the others appeared only after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Analysis with a cell sorter indicated no large scale meiotic changes in ploidy under the various culture conditions employed. These mutants may provide suitable, selectable, genetic markers for use in studying the possibility of a sexual mating process in Crithidia.

The aqueous degradation of dehydroascorbic acid (DHA) has been studied in the temperature range 52-90 degrees C. The DHA was determined by reversed-phase liquid chromatography and by derivatisation of DHA with o-phenylenediamine to form the fluorescent quinoxaline. The pseudo-first-order degradation of DHA has been verified and rate constants for the process are presented. The role of DHA in the degradation of ascorbic acid and previous DHA solution stability studies are discussed.

The relationship between bovine ocular squamous cell carcinoma and ultraviolet radiation was studied. Experimental procedures were devised to irradiate cattle with predetermined quantities of ultraviolet beta. Irradiation induced a preneoplastic ocular growth in one of four irradiated cattle. An epizootiologic study indicates that since 1950 the occurrence of bovine ocular squamous cel carcinoma reported at slaughter has increased. This increase was real and not due to an increase in numbers of cattle.

A vaccination study for infectious bovine keratoconjunctivitis was conducted on 108 newborn Hereford calves in the US Department of Agriculture Meat Animal Research Center cattle herd at Clay Center, Nebraska. Groups were allocated so that age of calf, sex of calf, and age of dam were equally distributed between the 54 vaccinated (group I) and the 54 nonvaccinated (group 2) control calves. The dams of both groups of calves were monitored as group 3 controls. An autogenous Moraxella bovis bacterin (formalin-killed, whole cells) was given IM at birth and at approximate intervals of 2 weeks for a total of 3 doses. Bacterial isolation rates for the cattle in groups 1, 2, and 3 during the summer were 92.6%, 92.6%, and 54.1%, respectively, and disease rates were 100%, 96.3%, and 70.6%. The rates were significantly (P less than 0.05) different between calves and cows. Vaccination of calves at birth permitted serum antibodies to develop before the calves were extensively exposed to infection; however, immunity to the disease did not develop. In a treatment study of other animals in the same herd, but in another pasture, the same criteria were used for allocation of 107 cow-calf pairs. Eye spray was applied to treated principals (group 4, 52 calves; and group 6, 53 cows) each week after examination and sample collection. Controls consisted of 54 calves (group 5) and 54 cows (group 7) that were examined and cultured bacteriologically in the same manner. The bacterial isolation and disease rates were less (P less than 0.05) in the treated calves (group 4) than in the nontreated controls (group 5). The differences in bacterial isolation rates between groups 6 and 7 were not significant, but group 6 had less (P less than 0.05) grade III lesions than did group 7. Weekly treatment appeared to be more effective in reducing the incidence of disease than did vaccination.

4-Nitroestrone 3-methyl ether has been shown to be an effective growth inhibitor of certain dimethylbenz(a)anthracene-induced rat mammary tumors in intact or ovariectomized rats. When administered at optimum levels (24 mg/kg daily), this A-ring-substituted estrone displayed no toxicity, slight estrogenicity, and an antitumor activity which was comparable to that of tamoxifen and nafoxidine and was surpassed only by ovariectomy or pharmacological doses of 17 beta-estradiol 3-benzoate. In addition, the appearance of mammary tumors was prevented when this estrogen derivative was administered to rats just prior to or after dimethylbenz(a)anthracene intubation. Unique to the action of the methyl ether of 4-nitroestrone on mammary tumors was the destruction of adenocarcinomas while permitting the appearance of fibroadenomas. Systemically, 4-nitroestrone 3-methyl ether brought about focal atrophy within the pituitary and ovaries while causing moderate hypertrophy of the uterus. Plasma prolactin was unaffected.