High-level expression of the 32.5-kilodalton calelectrin in ductal epithelia as revealed by immunocytochemistry

Abstract. Calelectrins are a family of antigenically related Ca²⁺-binding proteins that have only recently been described. They have the important property of binding to membranes only in the presence of Ca²⁺. We systematically studied the tissue localization of one calelectrin, the 32.5-kilodalton species, in rats using immunocytochemistry. We found that high levels were exclusively present in the epithelial cells of bile and pancreatic ducts, renal collecting ducts, bronchial epithelia, and brain ependyma. In all of these organs, the other cells were not immunoreactive. In addition, strong immunoreactivity was found in the intercalated disks of myocardial cells, and mild immunoreactivity was observed in several endocrine tissues. In contrast, the cellular distribution of the 67-kilodalton calelectrin was more diffuse, involving most parenchymal cells in addition to the already-mentioned cells. Due to the presence of high levels of 32.5-kilodalton calelectrin in some cell types, this protein may be used as a histochemical marker for differentiated ductal epithelial cells, some specialized epithelia, myocardial cells, and Paneth cells.     

Phenanthrene Biodegradation in Freshwater Environments

Phenanthrene, a low-molecular-weight polycyclic aromatic hydrocarbon, was incubated with water samples from various reservoir systems in Tennessee to evaluate the potential for significant polycyclic aromatic hydrocarbon degradation by the indigenous microbial populations. Biodegradation was assessed by comparison of total polycyclic aromatic hydrocarbon substrate recovery in degradation flasks relative to sterile control flasks. During 1977 field studies, the mean phenanthrene biodegradation was approximately 80% after a 4-week incubation. Within a given habitat, 45% of the total variability in phenanthrene biodegradation was attributable to the physical, chemical, and microbiological site characteristics examined. Polycyclic aromatic hydrocarbon degradation was directly related to the historical environmental pollution of the sampling sites examined, the length of biodegradation assessment, temperature, and the molecular size of the polycyclic aromatic hydrocarbon substrate.     

Thiobenzothiazole-modified Hydrocortisones Display Anti-inflammatory Activity with Reduced Impact on Islet β-Cell Function

Glucocorticoids signal through the glucocorticoid receptor (GR) and are administered clinically for a variety of situations, including inflammatory disorders, specific cancers, rheumatoid arthritis, and organ/tissue transplantation. However, glucocorticoid therapy is also associated with additional complications, including steroid-induced diabetes. We hypothesized that modification of the steroid backbone is one strategy to enhance the therapeutic potential of GR activation. Toward this goal, two commercially unavailable, thiobenzothiazole-containing derivatives of hydrocortisone (termed MS4 and MS6) were examined using 832/13 rat insulinoma cells as well as rodent and human islets. We found that MS4 had transrepression properties but lacked transactivation ability, whereas MS6 retained both transactivation and transrepression activities. In addition, MS4 and MS6 both displayed anti-inflammatory activity. Furthermore, MS4 displayed reduced impact on islet β-cell function in both rodent and human islets. Similar to dexamethasone, MS6 promoted adipocyte development in vitro, whereas MS4 did not. Moreover, neither MS4 nor MS6 activated the Pck1 (Pepck) gene in primary rat hepatocytes. We conclude that modification of the functional groups attached to the D-ring of the hydrocortisone steroid molecule produces compounds with altered structure-function GR agonist activity with decreased impact on insulin secretion and reduced adipogenic potential but with preservation of anti-inflammatory activity.     

Dexamethasone induces gelsolin synthesis and altered morphology in L929 cells

When L929 cells are exposed to 5 μg/ml dexamethasone, synthesis of a 90,000 M(r) polypeptide is induced within 12 h. Flattening of the cells begins at about this time and progresses to become quite prominent after 48 h of exposure. Two-dimensional PAGE and partial proteolytic fingerprints identify the 90,000 M(r) polypeptide as gelsolin, a Ca(++)-dependent inhibitor of actin polymerization. Thus, this system provides evidence that gelsolin may have a role in regulating cell shape in response to physiological agents such as glucocorticoids.     

Epratuzumab (humanised anti-CD22 antibody) in primary Sjögren's syndrome: an open-label phase I/II study

This open-label, phase I/II study investigated the safety and efficacy of epratuzumab, a humanised anti-CD22 monoclonal antibody, in the treatment of patients with active primary Sjögren's syndrome (pSS). Sixteen Caucasian patients (14 females/2 males, 33–72 years) were to receive 4 infusions of 360 mg/m² epratuzumab once every 2 weeks, with 6 months of follow-up. A composite endpoint involving the Schirmer-I test, unstimulated whole salivary flow, fatigue, erythrocyte sedimentation rate (ESR), and immunoglobulin G (IgG) was devised to provide a clinically meaningful assessment of response, defined as a ≥20% improvement in at least two of the aforementioned parameters, with ≥20% reduction in ESR and/or IgG considered as a single combined criterion. Fourteen patients received all infusions without significant reactions, 1 patient received 3, and another was discontinued due to a mild acute reaction after receiving a partial infusion. Three patients showed moderately elevated levels of Human anti-human (epratuzumab) antibody not associated with clinical manifestations. B-cell levels had mean reductions of 54% and 39% at 6 and 18 weeks, respectively, but T-cell levels, immunoglobulins, and routine safety laboratory tests did not change significantly. Fifty-three percent achieved a clinical response (at ≥20% improvement level) at 6 weeks, with 53%, 47%, and 67% responding at 10, 18, and 32 weeks, respectively. Approximately 40%–50% responded at the ≥30% level, while 10%–45% responded at the ≥50% level for 10–32 weeks. Additionally, statistically significant improvements were observed in fatigue, and patient and physician global assessments. Further, we determined that pSS patients have a CD22 over-expression in their peripheral B cells, which was downregulated by epratuzumab for at least 12 weeks after the therapy. Thus, epratuzumab appears to be a promising therapy in active pSS, suggesting that further studies be conducted.     

Extracorporeal bicarbonate space after bicarbonate or a bicarbonate-carbonate mixture in acidotic dogs

The effects of sodium bicarbonate and a bicarbonate-carbonate mixture on expired CO2 and the volume of distribution of bicarbonate were studied in eight anesthetized, paralyzed, and ventilated dogs made acidotic with HCl (5 mmol/kg) infused over 90 min. Both sodium bicarbonate and Carbicarb resulted in systemic alkalinization and comparable increases in the serum bicarbonate at 50 min (7.07 +/- 0.91 vs. 7.99 +/- 0.77, respectively; P = NS). Sodium bicarbonate infusion resulted in an increase in CO2 excretion that accounted for a fractional CO2 excretion of 0.20 +/- 0.09, whereas infusion of a bicarbonate-carbonate mixture resulted in a fractional CO2 excretion of -0.06 +/- 0.09 (P less than 0.01). The uncorrected volume of distribution of bicarbonate after sodium bicarbonate infusion was higher than that seen with the bicarbonate-carbonate mixture (0.60 +/- 0.07 vs. 0.34 +/- 0.03 l/kg; P less than 0.01). However, when the volume of bicarbonate distribution was corrected for expired CO2, there was no difference between treatment with sodium bicarbonate and the bicarbonate-carbonate mixture (0.44 +/- 0.07 vs. 0.38 +/- 0.04 l/kg; P = NS). These data demonstrate that, in this animal model of acidosis, sodium bicarbonate treatment of systemic acidosis is accompanied by a generation of a considerable amount of CO2, whereas treatment with a bicarbonate-carbonate mixture is not. This suggests that in states of impaired ventilation, a bicarbonate-carbonate mixture may offer more efficient systemic alkalinization and may be associated with less CO2 generation than sodium bicarbonate.     

G protein-coupled receptor (GPCR) kinase 2 regulates agonist-independent Gq/11 signaling from the mouse cytomegalovirus GPCR M33

The mouse cytomegalovirus M33 protein is highly homologous to mammalian G protein-coupled receptors (GPCRs) yet functions in an agonist-independent manner to activate a number of classical GPCR signal transduction pathways. M33 is functionally similar to the human cytomegalovirus-encoded US28 GPCR in its ability to induce inositol phosphate accumulation, activate NF-kappaB, and promote smooth muscle cell migration. This ability to promote cellular migration suggests a role for viral GPCRs like M33 in viral dissemination in vivo, and accordingly, M33 is required for efficient murine cytomegalovirus replication in the mouse. Although previous studies have identified several M33-induced signaling pathways, little is known regarding the membrane-proximal events involved in signaling and regulation of this receptor. In this study, we used recombinant retroviruses to express M33 in wild-type and Galpha(q/11)(-/-) mouse embryonic fibroblasts and show that M33 couples directly to the G(q/11) signaling pathway to induce high levels of total inositol phosphates in an agonist-independent manner. Our data also show that GRK2 is a potent regulator of M33-induced G(q/11) signaling through its ability to phosphorylate M33 and sequester Galpha(q/11) proteins. Taken together, the results from this study provide the first genetic evidence of a viral GPCR coupling to a specific G protein signaling pathway as well as identify the first viral GPCR to be regulated specifically by both the catalytic activity of the GRK2 kinase domain and the Galpha(q/11) binding activity of the GRK2 RH domain.