Microporosity of the substratum regulates differentiation of MDCK cells in vitro

Summary We have analyzed the ability of the physical substratum to modulate both the ultrastructural and protein synthetic characteristics of the Madin-Darby canine kidney (MDCK) renal cell line. When MDCK cells were seeded on Millipore Millicell CM microporous membrane cell culture inserts they demonstrated a more columnar organization with an increase in cell density sixfold greater than the same cells seeded on conventional plastic substrata. After 1 wk postseeding on the microporous membrane a partial basal lamina was noted, with a contiguous basement membrane being apparent after 2 wk. One-dimensional sodium dodecyl sulfate gel electrophoresis was used to analyze detergent-solubilized proteins from MDCK cells maintained on plastic substrata vs. microporous membranes. When proteins were pulse-labeled with [³⁵S]methionine, a 55 kDa protein was evident in the cytosolic extract of cells grown on collagen, laminin, and nontreated plastic substrata; but this labeled protein was not evident in similar extracts from cells grown on collagen and laminin-coated microporous membranes. To test if the polarized, basement-membrane secreting phenotype of the MDCK cells could be generated on a microporous membrane without pretreatment with any extracellular matrix (ECM) components, cells were seeded on the Millipore Millicell HA (cellulosic) microporous membrane. This type of substrata does not need a coating of ECM components for cell attachment. A partial basement membrane was formed below cells where the basal surface of the cell was planar, but not in areas where the cell formed large cytoplasmic extensions into the filter. This led us to the conclusion that the microporous nature of the substrata can dictate both ultrastructural and protein synthetic activities of MDCK cells. Furthermore, we suggest that both the planar nature of the basal surface and the microporosity of the substrate are corequisites for the deposition of the basement membrane.     

Ultrastuctural and cytochemical study of spermatogenesis in Scrobicularia plana (Mollusca,Bivalvia)

The ultrastructure of the mature spermatozoa and spermatogenesis of the bivalve Scrobicularia plana are described. Support cells extend from the basal lamina to the lumen of the testis and are laterally connected to the germinal epithelium. Germ cells present intercellular bridges and flagella since the spermatogonial stage. While spermatogonia and spermatocytes appear connected to support cells by desmosome-like junctions, elongated spermatids are held at the acrosomal region by support cell finger-like processes. During spermiogenesis, the acrosomal vesicle differentiates from a golgian saccule and then migrates to the nuclear apex. A microtubular manchette arising from centrioles surrounds the acrosomal vesicle, the nucleus, and the mitochondria at the time these three organelles start their elongation, disappearing after that. The mature spermatozoon of S. plana lacks a distinct midpiece because the mitochondria extend from the region of the pericentriolar complex along the nucleus anteriorly for approximately 1.4 μm. The features of this bivalve type of modified spermatozoon are compared with those of other animal groups having similar modifications.     

Lipid Peroxidation. Definition of Experimental Conditions for Selective Study of the Propagation and Termination Phases

To find experimental conditions to selectively study the propagation phase of lipoperoxidation we studied the lipoperoxidation, catalyzed by FeCl₂, of liposomes in a buffering condition where Fe²⁺ autoxidation and oxygen active species generation does not occur. Liposomes from egg yolk phosphatidylcholine. prepared by vortex mixing, do not oxidize Fe²⁺: on the contrary they oxidize Fe²⁺ when prepared by ultrasonic irradiation. Dimyristoyl phosphatidylcholine liposomes prepared by ultrasonic irradiation do not oxidize Fe²⁺. During sonication polyunsaturated fatty acid residues autoxidize and lipid hydroperoxides (LOOH) are generated. Only when LOOH are present in the liposimes Fe²⁺ oxidizes and its rate of oxidation depends on the amount of LOOH in the assay. The reaction results in the generation of both LOOH and thiobarbituric acid reactive material (TBAR): it is inhibited by butylated hydroxytoluene and has a acidic pH optimum; it is not inhibited by catalase and OH' scavengers. The reaction studied. thus, appears to be the chain branching and propagation phase of lipoperoxidation. When we studied the dependence of Fe²⁺ oxidation, LOOH and TBAR generation on FeCl₂ concentration, we observed that at high FeCl₂ concentrations the termination phase of lipoperoxidation was prevalent. Thus. by selecting the appropriate FeCl₂ concentration the proposed experimental system allows study of either the propagation or the termination phase of lipoperoxidation.     

Monoclonal antibody against a melanosomal protein in melanotic and amelanotic human melanoma cells

BALB/c mice were immunized with tyrosinase, partially purified in two stages from a human melanoma cell line. A hybridoma was obtained which produced monoclonal antibody (MoAb 1C11) reactive with 8/10 melanoma cell lines and 10/10 primary cultures of human melanocytes, neval cells, and melanomas. Immunoreactivity correlated to a certain extent with tyrosinase activity but not with melanin content. No crossreactivity was obtained with neuroblastoma, medulloblastoma, fibroblasts, keratinocytes, lymphoid cells, or murine melanomas. Purification of the antigen directly from cell lysates with a MoAb 1C11 CNBr-Sepharose affinity column gave a green-brown protein of 56 kDa with no detectable tyrosinase activity. This protein was therefore different from 60 kDa active tyrosinase, identified by enzyme activity and Western blotting with a MoAb derived previously (MoAb 5C12). Unlike 5C12, 1C11 reactivity was not destroyed by pretreatment of the antigen with periodate. Immunogold labelling showed that the 1C11-reactive antigen was associated with melanosomes, and there was close correlation between 5C12 and 1C11 reactivity in resistance to trypsin and in staining various melanocytic cell populations. MoAb 1C11 may therefore recognise a polypeptide epitope in a molecule closely linked to melanin biosynthesis.     

Differential distribution of antigen-specific helper T cells and cytotoxic T cells after antigenic stimulation in vivo. A functional study using limiting dilution analysis

We have developed modified limiting dilution analysis (LDA) techniques that distinguish in vivo Ag-stimulated murine helper T lymphocytes (HTL) and CTL from unstimulated precursor T cells, even those with the same Ag specificity. We refer to these cells that are detectable in the modified LDA as "Ag-conditioned" T cells (cHTL and cCTL). We have used the modified LDA techniques in conjunction with conventional LDA techniques (which enumerate all Ag-specific T cells) to evaluate the in vivo distribution of Ag-conditioned cHTL and cCTL following in vivo sensitization to alloantigens via sponge matrix or skin allografts. In general, we observed the following regarding the distribution of cHTL and cCTL: 1) Ag-conditioned HTL and CTL were detectable only after in vivo sensitization with alloantigen: 2) not all Ag-reactive T cells became conditioned T cells after in vivo Ag deposition; 3) the percentage of Ag-reactive T cells that converted to conditioned T cells after Ag deposition varied among different lymphoid compartments; 4) a high percentage of cHTL, but a low percentage of cCTL, accumulated in regional lymph nodes and spleen; 5) cHTL accumulated in peripheral blood, whereas cCTL did not; 6) Ag-conditioned cHTL were detectable in various lymphoid tissues for greater than 60 days following Ag deposition, whereas cCTL were detectable for only 14 to 20 days; and 7) unlike the other lymphoid sites, the site of Ag deposition accumulated a high percentage of both Ag-stimulated cHTL and cCTL. Furthermore, cHTL and cCTL appeared to reside in phenotypically distinct T cell subsets in that in vivo treatment with anti-L3T4 mAb abrogated the accumulation of HTL, but not CTL, at the site of Ag deposition. These data demonstrate differential compartmentalization of Ag-conditioned cHTL and cCTL subsequent to in vivo Ag deposition. The implications of these findings regarding the monitoring of in vivo immune responses are discussed.     

Apolipoprotein A-I metabolism in subjects with a PstI restriction fragment length polymorphism of the apoA-I gene and familial hypoalphalipoproteinemia

Familial hypoalphalipoproteinemia (hypoalpha), characterized by a decreased high density lipoprotein level, is associated with an increased incidence of premature cardiovascular disease. Restriction fragment length polymorphism analysis of genomic DNA has detected a polymorphism for the PstI restriction endonuclease near the apoA-I gene, with either a 2.2 or a 3.3 kb fragment. The latter has been previously found to occur with significantly higher frequency in probands of families with familial hypoalpha. ApoA-I was isolated from three unrelated subjects with familial hypoalpha and the 3.3 kb PstI polymorphism of the apoA-I gene, and from normal control subjects. The apoA-I from the hypoalpha subjects was structurally normal as determined by amino acid analysis and by two-dimensional gel electrophoresis. When normal apoA-I and hypoalpha apoA-I were simultaneously injected into either normal controls or hypoalpha subjects, both forms of apoA-I were catabolized at the same rate in the same subject, indicating that the hypoalpha apoA-I is also metabolically normal. Analysis of the kinetics of metabolism of apoA-I in the hypoalpha subjects, compared to the normal controls, revealed that the reduced plasma levels of apoA-I were due to an increased apoA-I fractional catabolic rate, and that the synthetic rate was normal. Based on these results, we conclude that the apoA-I gene in these hypoalpha subjects is normal, and the PstI polymorphism near the apoA-I gene, which is associated with familial hypoalpha, is likely to be a marker for a mutant gene closely linked to, but not in, the apoA-I gene.     

Intraspecific evolution of a gene family coding for urinary proteins

Summary The genome of the laboratory mouse contains about 35 major urinary protein (MUP) genes, many of which are clustered on chromosome 4. We have used distance and parsimony methods to estimate phylogenetic relationships between MUP genes from nucleotide sequence and restriction maps. By analyzing coding sequences we show that the genes fall into four main groups of related sequences (groups 1–4). Comparisons of restriction maps and the nucleotide sequences of hypervariable regions that lie 50 nucleotides 5 to the cap sites show that the group 1 genes and probably also the group 2 pseudogenes fall into subgroups. The most parsimonious trees are consistent with the evolution of the array of group 1 and 2 genes by mutation accompanied by a process tending toward homogenization such as unequal crossing-over or gene conversion. The phylogenetic grouping correlates with grouping according to aspects of function. The genomes of the inbred strains BALB/c and C57BL contain different MUP gene arrays that we take to be samples from the wild population of arrays.     

Notes on the use of wood refugia by Physella heterostropha (say) in a thermally disturbed swamp

Physella heterostropha size distributions and densities were measured in Pen Branch delta, a thermally altered swamp of the Savannah River, South Carolina. During cessation of thermal input, measurements were taken in a highly impacted area and more natural area. Of the three substrates sampled, logs, small woody material and benthos, snail density was highest on small pieces of submerged wood and < 20% of the snails on logs were out of the water. Following resumption of thermal input, snail density was four times higher on logs than previously measured. Snails responded to elevated water temperatures by congregating in a narrow band slightly above the water. Snail size did not appear to affect their response. However, the thermal history of the snails influenced their behaviour and survival rates. Logs may be a potential refuge for snails when water temperature are greater greater than 38°C. However, our results indicate that long-term survival in this manner may be impossible.