Hormonal regulation of estradiol biosynthesis, aromatase activity, and aromatase mRNA in rat ovarian follicles and corpora lutea

To determine the molecular basis for changes in aromatase (P450arom) activity in rat ovarian follicles and corpora lutea, seven clones for rat P450arom cDNA have been identified and isolated from a rat granulosa cell λgtll cDNA expression library using a 62 mer deoxyoligonucleotide probe (derived from an amino acid sequence of purified human placental aromatase) and a human placental P450arom cDNA probe. One of the rat P450arom cDNA clones contained an insert 1.2 kb in size. Both the human 1.8 kb cDNA and the rat 1.2 kb cDNA probes hybridized to a single species of P450arom mRNA that was 2.6 kb in size. Northern blot analysis revealed that corpora lutea isolated on day 15 of pregnancy contained high amounts of P450arom mRNA, whereas granulosa cells of antral follicles of hormonally primed, hypophysectomized rats (i.e., those from which mRNA was isolated to construct the cDNA library) contained only low amounts of P450arom mRNA. The lower amounts of P450arom in granulosa cells of preovulatory follicles in the estradiol-follicle-stimulating hormone primed hypophysectomized rats were unexpected because follicles incubated in medium containing testosterone substrate produce more estradiol than do corpora lutea isolated on day 15 of pregnancy and incubated under similar conditions. Additional studies will determine the hormonal events responsible for the elevated amounts and constitutive maintenance of P450arom mRNA and aromatase activity in luteal cells in vivo and in vitro.     

Critical role of the alpha 4 integrin/VCAM-1 pathway in cerebral leukocyte trafficking in lupus-prone MRL/fas(lpr) mice

MRL/fas(lpr) mice are affected by a systemic autoimmune disease that results in leukocyte recruitment to a wide range of vascular beds, including the cerebral microvasculature. The mechanisms responsible for the leukocyte trafficking to the brain in these animals are not known. Therefore, the aim of this study was to directly examine the cerebral microvasculature in MRL/fas(lpr) mice and determine the molecular mechanisms responsible for this leukocyte recruitment. Intravital microscopy was used to assess leukocyte-endothelial cell interactions (rolling, adhesion) in the pial microcirculation of MRL(+/+) (control) and MRL/fas(lpr) mice at 8, 12, and 16 wk of age. Leukocyte rolling and adhesion were rarely observed in MRL(+/+) mice of any age. MRL/fas(lpr) mice displayed similar results at 8 and 12 wk. However, at 16 wk, significant increases in leukocyte rolling and adhesion were observed in these mice. Histological analysis revealed that the interacting cells were exclusively mononuclear. Leukocyte rolling was reduced, but not eliminated in P-selectin(-/-)-MRL/fas(lpr) mice. However, leukocyte adhesion was not reduced in these mice, indicating that P-selectin-dependent rolling was not required for leukocyte recruitment to the cerebral vasculature in this model of systemic inflammation. E-selectin blockade also had no effect on leukocyte rolling. In contrast, blockade of either the alpha4 integrin or VCAM-1 eliminated P-selectin-independent leukocyte rolling. alpha4 Integrin blockade also significantly inhibited leukocyte adhesion. These studies demonstrate that the systemic inflammatory response that affects MRL/fas(lpr) mice results in leukocyte rolling and adhesion in the cerebral microcirculation, and that the alpha4 integrin/VCAM-1 pathway plays a central role in mediating these interactions.     

Amylase gene expression in intraspecific and interspecific somatic transformants of Drosophila.

The Amylase locus in Drosophila melanogaster normally contains two copies of the structural gene for alpha-amylase, a centromere-proximal copy, Amy-p, and a distal copy, Amy-d. Products of the two genes may display discrete electrophoretic mobilities, but many strains known to carry the Amy duplication are characterized by a single amylase electromorph, e.g., Oregon-R, which produces the mobility variant AMY-1. A transient expression assay was used in somatic transformation experiments to test the functional status of the Amy genes from an Oregon-R strain. Plasmid constructs containing either the proximal or distal copy were tested in amylase-null hosts. Both genes produced a functional AMY-1 isozyme. Constructs were tested against an AMY-3 reference activity produced by a coinjected plasmid that contains the Amy-d3 allele from a Canton-S strain. With reference to the internal control, the Amy-p and Amy-d genes from Oregon-R expressed different relative activity levels for AMY-1 in transient assays. The transient expression assay was successfully used to test the functional status of Amy-homologous sequences from strains of other species of Drosophila characterized by a single amylase elctromorph, namely, Drosophila pseudoobscura ST and Drosophila miranda S 204. The amylase-null strain of D. melanogaster provided the hosts for these interspecific somatic transformation experiments.     

Housing mice in a caging system with automatic flushing.

Male Har:(ICR)BR mice were housed 8 wk in wire bottom cages over paper, in wire bottom cages over an automatic cascade flushing system, or in solid-bottom plastic cages with bedding material. There were no substantial differences in general health or weight gain. Pentobarbital LD50 values were lower for the mice housed in wire bottom cages than for the mice in solid bottom cages with bedding. This difference was probably related to gastrointestinal content. It appears that the automatic cascade flushing system is suitable for housing mice for periods up to 8 wk.     

Metalloendopeptidases EC 3.4.24.15 and EC 3.4.24.16: potential roles in vascular physiology

Summary The zinc metalloendopeptidases EC 3.4.24.15 (EP 24.15) and EC 3.4.24.16 (EP 24.16) are closely related ubiquitous enzymes, which have well-defined in vitro activities in generation and degradation of a range of specific peptide targets. Despite this, little is known regarding their roles in whole animal physiology. One of the peptides degraded by these enzymes in vitro is bradykinin, a mediator with potent effects on the vasculature at both systemic and local levels. This review summarises the work that has examined the role of EP 24.15/24.16 in regulation of the vascular effects of bradykinin in vivo. This work was made possible by the development of a specific stable inhibitor of these enzymes, JA-2. Use of this inhibitor has shown that EP 24.15/24.16 are capable of regulating responses induced by exogenous bradykinin. This effect was observed at a systemic level with an increase in the hypotensive effect of intravenous bradykinin. Further work is required to determine whether these enzymes also regulate bradykinin produced endogenously.     

Degradation of 4,4'-dichlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl by the white rot fungus Phanerochaete chrysosporium.

The white rot fungus Phanerochaete chrysosporium has demonstrated abilities to degrade many xenobiotic chemicals. In this study, the degradation of three model polychlorinated biphenyl (PCB) congeners (4,4'-dichlorobiphenyl [DCB], 3,3',4,4'-tetrachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl) by P. chrysosporium in liquid culture was examined. After 28 days of incubation, 14C partitioning analysis indicated extensive degradation of DCB, including 11% mineralization. In contrast, there was negligible mineralization of the tetrachloro- or hexachlorobiphenyl and little evidence for any significant metabolism. With all of the model PCBs, a large fraction of the 14C was determined to be biomass bound. Results from a time course study done with 4,4'-[14C]DCB to examine 14C partitioning dynamics indicated that the biomass-bound 14C was likely attributable to nonspecific adsorption of the PCBs to the fungal hyphae. In a subsequent isotope trapping experiment, 4-chlorobenzoic acid and 4-chlorobenzyl alcohol were identified as metabolites produced from 4,4'-[14C]DCB. To the best of our knowledge, this the first report describing intermediates formed by P. chrysosporium during PCB degradation. Results from these experiments suggested similarities between P. chrysosporium and bacterial systems in terms of effects of congener chlorination degree and pattern on PCB metabolism and intermediates characteristic of the PCB degradation process.     

Energetics and regulations of formate and hydrogen metabolism by Methanobacterium formicicum

Accumulation of formate to millimolar levels was observed during the growth of Methanobacterium formicicum species on H₂–CO₂. Hydrogen was also produced during formate metabolism by M. formicicum. The amount of formate accumulated in the medium or the amount H₂ released in gas phase was influenced by the bicarbonate concentration. The formate hydrogenlyase system was constitutive but regulated by formate. When methanogenesis was inhibited by addition of 2-bromoethane sulfonate, M. formicicum synthesized formate from H₂ plus HCO _inf3 ^sup- or produced H₂ from formate to a steady-state level at which point the Gibbs free energy (G) available for formate synthesis or H₂ production was approximately -2 to -3 kJ/reaction. Formate conversion to methane was inhibited in the presence of high H₂ pressure. The relative rates of conversion of formate and H₂ were apparently controlled by the G available for formate synthesis, hydrogen production, methane production from formate and methane production from H₂. Results from ¹⁴C-tracer tests indicated that a rapid isotopic exchange between HCOO^- and HCO _inf3 ^sup- occurred during the growth of M. formicicum on H₂–CO₂. Data from metabolism of ¹⁴C-labelled formate to methane suggested that formate was initially split to H₂ and HCO _inf3 ^sup- and then subsequently converted to methane. When molybdate was replaced with tungstate in the growth media, the growth of M. formicicum strain MF on H₂–CO₂ was inhibited although production of methane was not Formate synthesis from H₂ was also inhibited.     

Targeting tumour necrosis factor alleviates signs and symptoms of inflammatory osteoarthritis of the knee

Introduction

Inflammation associated with synovial expression of TNFα is a recognised feature of osteoarthritis (OA), although no studies have yet reported beneficial effects of anti-TNFα therapy on clinical manifestations of inflammation in OA.

Methods

We conducted an open-label evaluation of adalimumab over 12 weeks in 20 patients with OA of the knee and evidence of effusion clinically. Inclusion criteria included daily knee pain for the month preceding study enrolment and a summed pain score of 125 to 400 mm visual analogue scale on the Western Ontario and McMaster University Osteoarthritis Index (WOMAC) pain subscale. The primary outcome was the Osteoarthritis Research Society International/Outcome Measures in Rheumatology Clinical Trials (OARSI/OMERACT) response criterion at week 12. Secondary outcomes included the WOMAC pain score 20% and 50% improvement, WOMAC stiffness and function scores, patient and physician global visual analogue scale, as well as target joint swelling.

Results

Treatment was well tolerated and completed by 17 patients with withdrawals unrelated to lack of efficacy or adverse events. By intention to treat, an OARSI/OMERACT response was recorded in 14 (70%) patients. WOMAC pain 20% and 50% responses were recorded in 14 (70%) patients and eight (40%) patients, respectively. Significant improvement was observed in mean WOMAC pain, stiffness, function, physician and patient global, as well as target joint swelling at 12 weeks (P < 0.0001 for all). After treatment discontinuation, 16 patients were available for assessment at 22 weeks and OARSI/OMERACT response compared with baseline was still evident in 10 (50%) patients.

Conclusion

Targeting TNFα may be of therapeutic benefit in OA and requires further evaluation in controlled trials.

Trial registration

ClinicalTrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT00686439","term_id":"NCT00686439"}}NCT00686439.     

Can erosions on MRI of the sacroiliac joints be reliably detected in patients with ankylosing spondylitis? - A cross-sectional study

Introduction

Erosions of the sacroiliac joints (SIJ) on pelvic radiographs of patients with ankylosing spondylitis (AS) are an important feature of the modified New York classification criteria. However, radiographic SIJ erosions are often difficult to identify. Recent studies have shown that erosions can be detected also on magnetic resonance imaging (MRI) of the SIJ early in the disease course before they can be seen on radiography. The goals of this study were to assess the reproducibility of erosion and related features, namely, extended erosion (EE) and backfill (BF) of excavated erosion, in the SIJ using a standardized MRI methodology.

Methods

Four readers independently assessed T1-weighted and short tau inversion recovery sequence (STIR) images of the SIJ from 30 AS patients and 30 controls (15 patients with non-specific back pain and 15 healthy volunteers) ≤45 years old. Erosions, EE, and BF were recorded according to standardized definitions. Reproducibility was assessed by percentage concordance among six possible reader pairs, kappa statistics (erosion as binary variable) and intraclass correlation coefficient (ICC) (erosion as sum score) for all readers jointly.

Results

SIJ erosions were detected in all AS patients and six controls by ≥2 readers. The median number of SIJ quadrants affected by erosion recorded by four readers in 30 AS patients was 8.6 in the iliac and 2.1 in the sacral joint portion (P < 0.0001). For all 60 subjects and for all four readers, the kappa value for erosion was 0.72, 0.73 for EE, and 0.63 for BF. ICC for erosion was 0.79, 0.72 for EE, and 0.55 for BF, respectively. For comparison, the kappa and ICC values for bone marrow edema were 0.61 and 0.93, respectively.

Conclusions

Erosions can be detected on MRI to a comparable degree of reliability as bone marrow edema despite the significant heterogeneity of their appearance on MRI.