Phylogenetic relationships of Blepharisma americanum and Colpoda inflata within the phylum ciliophora inferred from complete small subunit rRNA gene sequences

The complete small subunit rRNA gene sequences of the heterotrich Blepharisma americanum and the colpodid Colpoda inflata were determined to be 1719 and 1786 nucleotides respectively. The phylogeny produced by comparisons with other ciliates indicated that C. inflata is allied more closely with the nassophoreans and oligohymenophoreans than the spirotrichs. This is consistent with the placement of the colpodids in the Class Copodea. Blepharisma americanum was not grouped with the hypotrichs but instead was placed as the earliest branching ciliate. The distinct separation of B. americanum supports the elevation to class status given the heterotrichs based on morphological characters.     

Preliminary molecular studies on two chromosome 2 encoded genes, c-abl and beta 2M, in radiation-induced murine myeloid leukaemias

The majority of radiation-induced murine myeloid leukaemias are characterized by deletion and/or translocation of an interstitial region of chromosome 2, and there is evidence that such events may occur very early in myeloid leukaemogenesis. Analyses presented and discussed here on the structure and function of two possibly relevant chromosome 2 encoded genes (c-abl and beta 2M) lead to the preliminary conclusion that neither are directly involved nor activationally changed by the characteristic chromosome 2 rearrangements.     

Protein kinase C activity and reactivity to phorbol ester in vascular smooth muscle from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY)

Protein kinase C (PKC) activity in aortic and renal arterial smooth muscle from SHR (20-23 wk male; mean arterial pressure = 178 mm Hg) and WKY (age/sex matched; mean arterial pressure = 126 mm Hg) was quantitated. Activity was greatest in the particulate fractions relative to the soluble fractions in all sources. The only difference between SHR and WKY was in the soluble fraction from SHR renal arteries, which had 2 fold more activity (255 pmol/mg/min) when compared with WKY (136 pmol/mg/min). This difference was not apparently related to force modulation, since the magnitude of isometric force development in renal arteries in response to phorbol 12,13-dibutyrate was not different between SHR and WKY. The magnitude of force developed in response to phorbol 12,13-dibutyrate and PKC activity in the particulate fraction was greatest in aorta vs. renal arteries in both WKY and SHR. These results suggest that regional vascular differences in the amount of PKC activity may exist which are not apparently related to a disease state (i.e., hypertension). These differences may be related to differential sensitivity to phorbol ester-mediated contractions in isolated smooth muscle.     

Immunoelectron microscopic labeling of immunoglobulin in plasma cells after osmium fixation and epoxy embedding

Previous studies have found that immunoglobulin cannot be immunolabeled in tissues prepared for electron microscopy by usual methods. To test this conclusion, we used a protein A-gold postembedding immunolabeling method on tissues that were fixed in glutaraldehyde, post-fixed in osmium tetroxide, and embedded in epoxy resin; sections were pretreated with sodium metaperiodate. A variety of common fixation protocols were also used and the most suitable conditions for immunolabeling were determined. This technique permitted the ultrastructural localization of immunoglobulin light chains in optimally preserved and contrasted plasma cells from human tonsil, lymph nodes, plasmacytomas, and a renal biopsy. We were able to demonstrate multiple antigens in the same tissue and label antigens in tissues that had been stored for many years in epoxy resin. The technique allows quantitation of the gold label over plasma cell organelles and therefore gives information about the immunoglobulin secretory pathway in these cells. We found that the protein A-gold procedure compares favorably in technical ease with the immunoperoxidase, avidin-biotin peroxidase, and immunoglobulin-colloidal gold immunolabeling methods, and has added advantages in allowing precise localization and quantitation of the labeled antigen.     

Cleavage of human high molecular weight kininogen by factor XIa in vitro. Effect on structure and function

We have recently demonstrated that human high molecular weight kininogen (HMWK) is a pro-cofactor that is cleaved by kallikrein to yield a two-chain cofactor (HMWKa) and the nanopeptide bradykinin. This proteolysis enhances its association with an activating surface, an event necessary for expression of its cofactor activity. We now report that factor XIa is capable of hydrolyzing HMWK and releasing bradykinin in a purified system as well as cleaving and inactivating HMWK in a plasma environment during the contact-activation process. The profile of proteolysis differs from that produced by kallikrein and by factor XIIa in that the first cleavage by factor XIa yields 75- and 45-kDa polypeptides, whereas both factor XIIa and kallikrein initially produce 65- and 56-kDa species. Further proteolysis by all three enzymes eventually produces similar heavy chains (Mr = 65,000) and light chains (Mr = 45,000). However, the amount of factor XIa generated in plasma during contact activation further degrades the light chain of HMWK, eventually destroying its coagulant activity. Furthermore, in a purified system, enhancement of the degradation of HMWK coagulant activity by factor XIa was achieved when kallikrein was included in the incubation mixture, suggesting that the preferred substrate for factor XIa is the active form of HMWK (HMWKa), and not the pro-cofactor. These data suggest that factor XIa has the potential to act as a regulator of contact-activated coagulation by virtue of its ability to destroy the cofactor function of HMWK after its generation by either kallikrein, factor XIIa, or to a lesser extent, factor XIa, itself.     

Identification of a tumor inhibitory factor in rat ascites fluid

A polypeptide which inhibits the growth of human carcinoma cells has been characterized from Novikoff rat ascites fluid. This tumor inhibitory factor co-purified with transforming growth factor activity through acid/ethanol extraction and Bio-Gel chromatography. The two activities were completely separated by reverse phase HPLC. The tumor inhibitory factor is heat stable and requires disulfide bonds for bioactivity. This factor inhibited the anchorage independent growth of the more differentiated human colon carcinoma cell lines but did not affect the less differentiated carcinoma cells. The presence of stimulatory and inhibitory activities in the same extracts suggests that the relative concentrations of these factors may be important in the control of cell growth.     

NOTES ON THE ECOLOGY OF A SPECIES OF ZYGOGONIUM (KÜTZ.) IN YELLOWSTONE NATIONAL PARK

A species of Zygogonium forms extensive dark purple mats in Yellowstone National Park in acidic habitats adjacent to thermal areas. These mats range up to 6 cm in thickness and up to 3000 m² in areal extent. Temperatures in the mats varied from 20–31 C and pH varied from 2.4–3.1. These mats form on soil in areas where a moist surface is created by the presence of small acidic springs or seeps. The effect of light, temperature, and pH on photosynthesis was studied in the field by use of ¹⁴CO₂. Photosynthesis increased in rate up to full sunlight; light inhibition was not observed. Temperature optimum for photo-synthesis was 25 C. A broad pH optimum was found between 1.0 and 5.0. The Zygogonium mats have a high water holding capacity and create a moist habitat in which Euglena and Chlamydomonas develop. The mats also serve as repositories for the eggs of the brine fly Ephydra bruesi and both larvae and adults of this fly probably consume Zygogonium filaments as their main food source.     

A premature acrosome reaction is programmed by mouse t haplotypes during sperm differentiation and could play a role in transmission ratio distortion

Mouse t haplotypes are variant forms of chromosome 17 that can be transmitted at non-Mendelian ratios by heterozygous +/t males. The accumulated genetic data indicate that '+-sperm' and 't-sperm' are produced in equal numbers but that most '+-sperm' are rendered dysfunctional, so that 't-sperm' have a relative advantage at fertilization. To date, the basis for this t-induced sperm dysfunction has remained unknown. Here we demonstrate that a high proportion of sperm obtained from certain strains of +/t mice undergo a premature acrosome reaction under in vitro capacitation conditions. The simplest interpretation of these data, in conjunction with previous results, is that developing '+-spermatids' are preprogrammed by 't-spermatids' to undergo this premature reaction. Since acrosome-reacted sperm are unable to participate in the process of fertilization, this defect could account for the extreme distortion of transmission ratio observed from mice heterozygous for a class of complete t haplotypes.