Isolation and purification of a squamous cell carcinoma of the head and neck-associated antigen identified by autologous antibody

We have previously shown that detection of autologous antibody activity to squamous cell carcinoma of the head and neck may be augmented by dissociation of immune complexes. Western blot analysis with autologous antibody has identified a 60 kDa squamous cell carcinoma of the head and neck-associated antigen in spent media and immune complex-dissociated serum ultrafiltrate not recognized by normal human sera. Antigen-containing fractions of spent media were eluted from anion exchange columns immediately after serum albumin indicating that the antigen has an acidic pI < 4. Preparative purification of the squamous cell carcinoma antigen was accomplished by anion exchange of concentrated spent media (protein concentration 300 mg/ml) followed by lectin affinity chromatography with a Triticum vulgaris column. A single 60 kDa band was detected by silver stain and Western blot in antigen-containing fractions eluted following lectin affinity chromatography and SDS-PAGE. Final concentration of the antigen was determined to be 1 microgram/ml of protein with relative activity increased 1600 x over unfractionated spent media. We conclude that a squamous cell carcinoma of the head and neck-associated antigen, detected by autologous antibody, is an acidic 60 kDa glycoprotein.     

Recombinant entactin promotes mouse primary trophoblast cell adhesion and migration through the Arg-Gly-Asp (RGD) recognition sequence

In vitro culture of mouse blastocysts during the period coinciding with implantation has revealed that primary trophoblast cells can adhere and migrate in serum-free medium when provided with certain extracellular matrix components, including fibronectin and laminin. Tightly associated with laminin is the glycoprotein, entactin, that may play an important role in basement membrane assembly and cell attachment. Mouse blastocysts were studied using this in vitro model to determine whether entactin was capable of mediating trophoblast invasive activity. Although entactin has never been shown to promote cell migration, we report here that recombinant entactin supported blastocyst outgrowth in a dose-dependent manner, with a maximal effect at 20-50 micrograms/ml. The ability of trophoblast cells to adhere and migrate on entactin was specifically inhibited by anti-entactin antibody, but not by antibodies raised against laminin. The synthetic peptide, Gly-Arg-Gly-Asp-Ser-Pro, that contains the Arg-Gly-Asp (RGD) integrin recognition site, reversibly inhibited entactin-mediated blastocyst outgrowth in a dose-dependent manner, but had no effect on laminin-mediated outgrowth. The synthetic peptide, Gly-Phe-Arg-Gly-Asp-Gly-Gln, that comprises the actual RGD-containing sequence within entactin, promoted trophoblast outgrowth when immobilized on the substratum. Furthermore, a mutated recombinant entactin, altered to contain a Glu in place of Asp at the RGD site, provided no trophoblast cell adhesive activity. We conclude that entactin promotes trophoblast outgrowth through a mechanism mediated by the RGD recognition site, and that it may play an important role during invasion of the endometrial basement membrane at implantation.     

FTIR spectroscopy study of PTHrP(1-34) involved in humoral hypercalcaemia of malignancy

The components of secondary structure of the biologically-active N-terminal domain of human parathyroid-hormone-related protein (residues 1-34) and several truncated species were examined using Fourier transform infrared (FTIR) spectroscopy. The major structural features include a segment of alpha-helix within the N-terminal segment probably extending from Glu-4 to Lys-11 with three beta-turns localized to the segments Gly-12 to Ile-15, Gln-16 to Arg-20 and His-25 to Ala-29. Some beta-sheet was detected in the full-length peptide, but not in any of the C-terminal truncated samples. These structural features were studied in the smaller peptides for the purpose of localization of the various components and with a view to describing the region likely to form the bulk of the receptor binding site.     

Cubital tunnel syndrome and the painful upper extremity

Cubital tunnel syndrome is the second-most-common compressive neuropathy. With the increasing prevalence of entrapment neuropathies, the presentation of ulnar nerve compression with a painful upper extremity appears to be more common. Although our knowledge and understanding of this disease are increasing, the principles of management remain constant. We are obliged to reach a timely and appropriate diagnosis to minimize the extent of neurologic injury and institute an appropriate treatment regimen to preserve and restore normal neural function. Although there are many ways to reach these goals, the avoidance of complications is paramount to achieve a reliable and pain-free outcome. Preventing injury to the medial antebrachial cutaneous nerve, complete release of all sites of compression, and avoidance of creating new compressive sites are the keys to this end.     

Improving neglected influenza vaccination among healthcare workers in long-term care

OBJECTIVE: To increase utilization of influenza vaccination among long-term-care health workers. DESIGN: Before-after intervention study. SETTING: A 300-bed life-care community served by 195 staff employees. INTERVENTION: An educational intervention followed by a Vaccination Fair. MEASUREMENTS: Percent receiving vaccination. A post-intervention survey was used to define staff attitudes towards vaccination. RESULTS: Use of influenza vaccination among staff increased from 8% the previous year to 46% during a Vaccination Fair and to 54% during a similar Fair the next year. CONCLUSIONS: Organized institutional efforts to promote immunization among health-care workers, such as a Vaccination Fair, improves compliance among health-care workers.     

Induction of cytochrome P450 and peroxisomal enzymes by clofibric acid in vivo and in vitro

We have analysed the induction of microsomal and peroxisomal proteins and their RNAs after treatment of hepatocytes with the peroxisome proliferator, clofibric acid, in vitro and in vivo. After treatment of hepatocytes with 1 mM clofibric acid for 4 days, P450 4A1 RNA is induced 500-fold, and acyl-CoA oxidase and P450 2B1 280-fold, relative to control cultures. These RNAs are detectably induced after administration of 25 microM clofibric acid, and show a similar induction response with increasing doses of clofibric acid. Western blot analysis of the P450 4A and bifunctional enzyme (BFE) proteins showed that both were induced in parallel with increasing doses of clofibric acid, over a range of 25 microM-1 mM. The distribution of the induced proteins was examined by immunocytochemistry. Increasing doses of clofibric acid led to an increase in the average intensity of staining for both proteins throughout the hepatocyte population. There was, however, a graded variation between hepatocytes in the intensity of staining, both for P450 4A and BFE proteins. The heterogeneity in response of the hepatocyte population in vitro may be related to differential sensitivity of hepatocytes to induction in vivo. Therefore, rats were dosed with 0, 50 or 300 mg/kg of clofibric acid for 4 days by gavage, and the livers were examined by immunocytochemistry. After 50 mg/kg of clofibric acid, both P450 4A and BFE were induced mainly in zones 3 and 2 of the liver acinus. However, after 300 mg/kg of clofibric acid, staining for both proteins was strong and homogenous throughout the liver acinus. Thus, hepatocytes from zones 3 and 2 of the acinus are differentially responsive to induction by clofibric acid.     

Origins of polymorphism at a polypurine hypervariable locus

We present characterisation of a hypervariable locus, D8S210, mapped to the telomeric region of the short arm of chromosome 8. The locus is highly polymorphic with alleles varying in size from 1.8 kb to 24 kb. Sequence data from 7 alleles shows that the variable region is entirely polypurine on one strand with a tetranucleotide repeating unit GGAA at the margins and diverged versions of this motif internally. The margins are conserved between alleles; polymorphism occurring in the internal regions of the repeat. Alleles are inherited in a Mendelian manner and one new mutation has been observed in analysis of 51 meioses. Use of single copy flanking sequences to elaborate the polymorphism revealed loss of single copy DNA in 3 unrelated families and in 2 other unrelated individuals. Restriction mapping shows that this loss is similar for different sized alleles in all three families suggesting that it was an early event that may have involved a flanking Alu sequence. We present evidence that the polypurine region can adopt triplex conformations in vitro. Such structures may facilitate loss or gain of unique sequences in the genome, contribute to mutation at conformation transition points and drive the hypervariability (> 99% heterozygosity) of this locus.