The temperature-dependent stoichiometry of mixed cryoimmunoglobulins

The interaction of three monoclonal rheumatoid factor IgM molecules with IgG antigens has been studied utilizing immunoglobulins isolated from three mixed cryoglobulins. Static light scattering measurements show that the stoichiometry of these immune complexes changes in a temperature-dependent manner from IgM(IgG)0-2 at temperatures greater than 37 degrees C to IgM(IgG)5 complexes at temperatures below 15 degrees C. These results were confirmed by the analysis of the composition of polyethyleneglycol-precipitated complexes. For one mixed cryoglobulin (Glo), temperature-dependent changes in stoichiometry were also verified by chemical cross-linking studies. Binding constants were determined by Scatchard analysis of light scattering data and by fluorescence polarization measurements. Values on the order of 10(5) M-1 were obtained for three monoclonal rheumatoid factor IgM molecules. Glo was further investigated by dynamic light scattering and partial specific volume measurements. Both dynamic light scattering and partial specific volume measurements provided evidence for surprising shape changes of the IgM X IgG complex as a function of temperature and IgG stoichiometry. Collectively, the data support the simple hypothesis that cryoprecipitation of mixed cryoglobulins occurs as a consequence of increases in the size (stoichiometry) of the complexes that are formed at low temperatures.     

AKAP350 at the Golgi apparatus. I. Identification of a distinct Golgi apparatus targeting motif in AKAP350

The protein kinase A-anchoring proteins (AKAPs) are defined by their ability to scaffold protein kinase A to specific subcellular compartments. Each of the AKAP family members utilizes unique targeting domains specific for a particular subcellular compartment. AKAP350 is a multiply spliced AKAP family member localized to the centrosome and the Golgi apparatus. Three splicing events in the carboxyl terminus of AKAP350 generate the AKAP350A, AKAP350B, and AKAP350C proteins. A monoclonal antibody recognizing all three splice variants as well as a polyclonal antibody specific for AKAP350A demonstrated both centrosomal and Golgi apparatus staining in paraformaldehyde-fixed HCA-7 cells. Golgi apparatus-associated AKAP350A staining was dispersed following brefeldin A treatment. Using GFP chimeric constructs of the carboxyl-terminal regions of AKAP350A, a Golgi apparatus targeting domain was identified between amino acids 3259 and 3307 of AKAP350A. This domain was functionally distinguishable from the recently described centrosomal targeting domain (PACT domain, amino acids 3308-3324) located adjacent to the Golgi targeting domain. These data definitively establish the specific association of AKAP350A with the Golgi apparatus in HCA-7 cells.     

The role of ADAM 15 in glomerular mesangial cell migration

Mesangial cells (MC) occupy the core of the renal glomerulus and are surrounded by a mesangial matrix. In certain diseases, MC migrate through this matrix into the pericapillary space. The mechanisms involved, however, are poorly understood. Members of the ADAM (A Disintegrin And Metalloproteinase) family of membrane proteins have the potential to be key modulators of cell-matrix interactions through the activities of their constituent domains. We have studied the possible role of ADAM 15 in human (H) MC migration in vitro. HMC ADAM 15 was expressed at low levels in serum-free medium but was increased during migration. Antibodies to the individual domains of ADAM 15 and the incorporation of antisense ADAM 15, (but not control oligonucleotide) inhibited this migration. Furthermore, inhibition of migration by the broad spectrum metalloproteinase inhibitor BB3103, demonstrated that metalloproteinase activity was essential for migration. ADAM 15, extracted from HMC membranes, was an active metalloproteinase, which degraded both type IV collagen and gelatin prepared from fibrillar collagen. Activity was inhibited by EDTA but not by phenylmethylsulfonyl fluoride. This is the first report of the potential of ADAM 15 for involvement in the restructuring of the mesangial matrix and in the migration of MC in disease.     

Bioarcheology of the South Central United States

Bioarcheology of the South Central United States. Edited by Jerome C. Rose. Fayetteville, AR: Arkansas Archeological Survey Research Series No. 55. 1999. 297 pp. ISBN 1‐56349‐086‐2. $30.00 (paper).     

A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition

Background  

Real-time PCR analysis is a sensitive DNA quantification technique that has recently gained considerable attention in biotechnology, microbiology and molecular diagnostics. Although, the cycle-threshold (Ct) method is the present "gold standard", it is far from being a standard assay. Uniform reaction efficiency among samples is the most important assumption of this method. Nevertheless, some authors have reported that it may not be correct and a slight PCR efficiency decrease of about 4% could result in an error of up to 400% using the Ct method. This reaction efficiency decrease may be caused by inhibiting agents used during nucleic acid extraction or copurified from the biological sample.     

TGF-beta1-mediated fibroblast-myofibroblast terminal differentiation-the role of Smad proteins

It is now clear that resident myofibroblasts play a central role in the mediation of tissue fibrosis. The aim of the work outlined in this study is to increase our understanding of the mechanisms which drive the phenotypic and functional changes associated with the differentiation process. We have used an in vitro model of transforming growth factor-beta1 (TGF-beta1)-induced pulmonary fibroblast-myofibroblast differentiation to examine the role of the TGF-beta1 Smad protein signaling intermediates, in alterations of fibroblast phenotype and function associated with terminal differentiation. TGF-beta1 induced marked alteration in cell phenotype, such that cells resembled "epithelioid-postmitotic fibroblasts." This was associated with marked reorganization of the actin cytoskeleton and upregulation of alphaSMA gene expression. TGF-beta1 stimulation also induced alphaSMA protein expression with increased incorporation of alphaSMA into stress fibers. Following stimulation with TGF-beta1, subsequent addition of serum-free medium did not reverse TGF-beta1-induced morphological change, suggesting that TGF-beta1 induced a relatively stable alteration in fibroblast cell phenotype. Functionally, these phenotypic changes were associated with induction of type I, type III, and type IV collagen gene expression and an increase in the concentrations of the respective collagens in the cell culture supernatant. The role of Smad proteins in terminal differentiation of fibroblasts was examined by transfection of cells, with expression vectors for the TGFbeta1 receptor-regulated Smads (R-Smads) or the co-Smad, Smad 4. Transfection with Smad2 but not Smad3 resulted in TGF-beta1 independent alteration in fibroblast cell phenotype, up-regulation of alphaSMA mRNA and reorganization of the actin cytoskeleton. Transfection with Smad4 also induced alteration in cell phenotype, although this was not as pronounced as the effect of overexpression of Smad2. Overexpression of the Smad2, Smad3, or Smad4 proteins was associated with increased production of all collagen types. The study suggests that the phenotypic and functional changes associated with TGF-beta1-induced fibroblast terminal differentiation are differentially regulated by Smad proteins.     

A review of the Pterogeniidae (Goleoptera)

The Pterogeniidae, a family of beetles from Indoaustralia, are revised. They comprise five genera and 24 species. Three genera and 17 species are described as new and one species is synonymized. It is shown that the male and particularly the female genitalia provide useful means for species definition. The phylogenetic relationships are discussed based on a cladistic analysis of 23 morphological characters using PAUP. The analysis resulted in a single cladogram with following grouping: ( Kryptogenius + ( Tychogenius + ( Katagenius + ( Plerogenins + Histanocerus )))). For rooting the cladogram and polarizing the characters, Sivacrypticus indicus (Archeocrypticidae) was used as an outgroup. The majority of the species is restricted to insular tropical Asia and Oceania but four of them extend their range onto the Malayan Peninsula. Another four species are known only from continental Asia, i.e. two species from South India and one each from Malayan Peninsula and Vietnam respectively. Species of Kryptogenius, Pterogenius, Katagenius and Tychogenius are highly endemic and could therefore potentially be useful for analysing areas of endemism. For this, however, the cladistic relationships should be resolved at species level. Species of Histanocerus are more widely distributed but none is found on both sides of Wallace's line.     

Comparison of carpal canal pressure in paraplegic and nonparaplegic subjects: clinical implications

The purpose of this study was to evaluate the pressure within the carpal tunnel that was generated with certain tasks in paraplegic versus nonparaplegic subjects. Four groups of subjects were evaluated: 10 wrists in six paraplegic subjects with carpal tunnel syndrome, 11 wrists in six paraplegics without the syndrome, 12 wrists in nine nonparaplegics with the syndrome, and 17 wrists in 11 nonparaplegics without the syndrome. Carpal canal pressures were measured in the wrists in three positions (neutral, 45-degree flexion, 45-degree extension) and during two dynamic tasks [wheelchair propulsion and RAISE (relief of anatomic ischial skin embarrassment) maneuver]. External force resistors were placed over the carpal canal and correlated with internal tunnel pressures. At each wrist position, paraplegics with carpal tunnel syndrome consistently had higher carpal canal pressure than did the other groups at the corresponding wrist position; statistical significance was evident with regard to the neutral wrist position (p < 0.05). Within each group of subjects, wrist extension and wrist flexion produced a statistically significant increase in carpal canal pressure (p < 0.05), compared with the neutral wrist position. Dynamic tasks (wheelchair propulsion and the RAISE maneuver) significantly elevated the carpal canal pressure in paraplegics with carpal tunnel syndrome, compared with the other groups (p < 0.05). Lastly, there is a linear positive correlation between carpal canal pressure and external force resistance.