The regulation of intermediate filament reorganization in mitosis. p34cdc2 phosphorylates vimentin at a unique N-terminal site

The disassembly of vimentin-containing intermediate filament (IF) networks during mitosis in BHK-21 cells is accompanied by increased phosphorylation of vimentin (Chou, Y.-H., Rosevear, E., and Goldman, R. D. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1885-1889). We have recently identified p34cdc2 as the catalytic subunit of one of the two endogenous vimentin kinases in mitotic baby hamster kidney cells (Chou, Y.-H., Bischoff, J. R., Beach, D., and Goldman, R. D. (1990) Cell 62, 1063-1071). To begin to characterize the biochemical basis of the p34cdc2-mediated IF disassembly process, we have purified and sequenced the 32P-labeled tryptic peptides derived from in vitro-phosphorylated vimentin. The results demonstrate that Ser-55, in the N-terminal non-alpha-helical domain of vimentin, is the most favored phosphorylation site. This finding supports the idea that the N-terminal domain of type III IF protein plays a crucial role in regulating IF structure and supramolecular organization.     

Structural basis of a conserved idiotope expressed by an autoreactive human B cell lymphoma. Evidence that a VH CDR3 mutation alters idiotypy and specificity

Our laboratory has previously investigated the relationship of autoimmune disease and B cell neoplasia in a patient with a diffuse, well differentiated splenic B cell lymphoma and associated autoimmune hemolysis due to an anti-Pr2 antibody. EBV-immortalized B cell clones, established from this lymphoma, were shown to secrete the same pathologic anti-Pr2 antibody. The antiidiotypic mAb, RI.1, defined a private Id (IdRI.1) of the anti-Pr2 antibody that was related to the Ag-binding site and was expressed by both the lymphoma and derived cell lines. This unique Id was expressed by the majority of splenic tumor B cells and also was conserved over a period of 4 yr. In this report, the structural basis of IdRI.1 expression was investigated by analysis of Id- variants isolated by flow microfluorimetry using RI.1. Six Id- cell lines that secrete IgM kappa but lack Pr2 specificity were generated from an Id+ cell line, LS2. These lines were shown to be related to LS2 and the lymphoma by karyotype and by restriction fragment analysis of Ig gene rearrangements. Shared and unshared nucleotide substitutions in the VH and VL regions of the six independent clones were used to construct a genealogic tree relating the Id- clonal members to a common Id+ precursor. The tree illustrates that the base changes occurred sequentially, suggesting that they were introduced by somatic point mutation. Only one VH CDR3 bp difference from the LS2 nucleic acid sequence is common to all Id- sequences, resulting in an amino acid substitution of cysteine 108 to tyrosine. Taken together, these findings suggest that both the expression of IdRI.1 and Ag binding are affected by a single mutation localized to the D region of the anti-Pr2 antibody.     

Trend,seasonality, cycle,and irregular fluctuations in primary productivity at Lake Tahoe,California-Nevada,USA

Primary productivity has been measured routinely at Lake Tahoe since 1967, and a number of mechanisms underlying variability in the productivity record have now been identified. A long-term trend due to nutrient loading dominates the series. Seasonality also is prominent, apparently controlled by direct physical factors unrelated to the trophic cascade. A 3-yr cycle has been detected and several possible mechanisms are considered. Irregular fluctuations also are present, caused in part by isolated events (a forest fire) and recurring but variable phenomena (spring mixing). Except possibly for the 3-yr cycle, the known sources of variability appear to operate bottom-up through direct physical and chemical effects on the phytoplankton.     

Phagotrophy and NH4+ regeneration in a three-member microbial food loop

In a series of batch experiments we compared the efficiencyof nitrogen regeneration of a two- and three-member microbialfood loop consisting of a mixed bacterial assemblage, a small(3–5 µm) heterotrophic flagellate (Paraphysomonassp.), and a large (7–12 µm) heterotrophic flagellate(Paraphysomonas imperforata). In the two-member system the nitrogenregeneration efficiency for NH₄⁺ (R_n) was 41% and the grossgrowth efficiency (GGE) was 57% during active grazing by thesmall flagellate on bacteria. Regeneration of NH₄⁺ continuedduring the stationary phase so that R_n was 75% after 6 daysincubation. When the larger flagellate was introduced at theend of exponential growth of the smaller grazer in the three-membersystem, initially there was rapid regrowth of bacteria, tyingup 15% of the nitrogen originally in the bacteria. The largerflagellate grazed the smaller one with a GGE of 55%. Total nitrogenregeneration efficiency through exponential growth of the largerflagellate was 73%. Because microbial food loops in naturalwaters are far more complicated and with more grazing stepsthan portrayed in this study, we would expect the bulk of nutrientswithin these systems to be recycled with little transfer tohigher trophic levels.     

Vimentin is hyperphosphorylated in primary human fibroblasts treated with okadaic acid.

Okadaic acid and dinophysistoxin-1 (35-methylokadaic acid) induced hyperphosphorylation of a 58 kDa protein in primary human fibroblasts, due to inhibition of protein phosphatase 1 and 2A activities. The protein was present in the nuclear and cytosolic fractions. Its pI was 5.3. The hyperphosphorylated protein reacted with monoclonal and polyclonal anti-vimentin antibodies, but not with anti-nucleolin antibody. Phosphorylation of vimentin was stimulated in vitro by dinophysistoxin-1 dose-dependently in the presence of protein phosphatase 2A and protein kinases.     

Linkage mapping of human polymorphic proteins identified by two-dimensional electrophoresis.

Nineteen polymorphic lymphocyte proteins were previously detected by two-dimensional protein electrophoresis (2DE). In this report, we describe the genetic linkage mapping of six of these polymorphic proteins (PNIA1-PNIA6), the identification by genetic linkage of a seventh (glyoxalase 1 on 6p21), and support for the mapping of an eighth (plastin or LCP1) to near the ESD locus on Chr 13. PNIA1-PNIA6 were assigned, respectively, to 10q26, 16p13.3, 10q, 11p15, 3q, and 19q13. These genetic linkages were achieved by classical linkage analysis of 2DE protein charge polymorphisms to the panel of RFLPs previously typed in nine pedigrees in the Centre D'Etude du Polymorphisme Humain (CEPH) collection.     

Induction of adult-type nicotinic acetylcholine receptor gene expression in noninnervated regenerating muscle

Expression of adult-type nicotinic acetylcholine receptors at the neuromuscular junction is thought to result from selective induction of their genes in endplate-associated nuclei due to local neurotrophic control. However, denervation studies indicate that endplate-specific expression can be maintained in the absence of the nerve. We investigated the role played by the basal lamina in this expression by assaying for the adult-type-specific epsilon RNA in noninnervated regenerating muscle. We found that this RNA is locally expressed beneath the old endplates after 10 days of regeneration. At earlier times epsilon RNA is also found in areas other than the endplate region. These results indicate that in adult muscle the basal lamina contains all the components necessary to direct nicotinic acetylcholine receptor gene expression to the endplate.     

Effects of subcultivation and culture medium on differentiation of human fetal cardiac myocytes

Summary Previous work has suggested that subcultivated human fetal heart muscle cell cultures contain immature cardiac muscle cells capable only of limited differentiation after mitogen withdrawal. We studied several human fetal heart cultures (14–15 wk gestation) at several passage levels using immunocytochemistry, autoradiography, and Northern blot analysis. Characteristics in high-mitogen (growth) medium were compared with those after serum withdrawal. Cultured cells from one heart, expanded through 2 passages in growth medium, did not beat; however, 75% of cells did beat after subsequent culture for 24 days in low-serum (differentiation) medium containing insulin. In confluent cultures after 1 passage, there was no detectable difference in the number of cardiac myocytes present in growth medium compared with that 7 days after serum withdrawal. After 4 passages, however, serum withdrawal increased the number of cells expressing immunoreactive sarcomeric myosin heavy chain by 100-fold; expression of immunoreactive sarcomeric actin andα-cardiac actin mRNA also increased in the same cultures. Similar results were obtained in cultures kept in differentiation medium for 20 days before passage and expansion in growth medium. Using isopycinc centrifugation, a high-density cell fraction was isolated which contained no immunostained myocytes in growth medium but numerous myocytes after serum withdrawal. Combined immunocytochemistry/autoradiography showed that myocytes synthesize DNA in growth medium and in serum-free medium containing fibroblast growth factor, but not in serum-free medium alone. The results indicate that a) human fetal cardiac muscle cells proliferate in vitro and can maintain a phenotype characteristic of fetal myocytes after multiple subcultivations followed by serum withdrawal; b) after subcultivation in growth medium, some myocytes modulate their phenotype into one in which detectable levels of cardiac contractile proteins are expressed only after mitogen withdrawal, and c) the phenotype attained after serum withdrawal is in part dependent on passage level. Cultured human fetal myocardial cells my provide a useful experimental system for the study of human cardiac muscle cell biology.     

Reactive oxygen species and elastase mediate lung permeability after acid aspiration.

Acid aspiration leads to increased neutrophil (PMN) oxidative metabolism, an event associated with lung leukosequestration and permeability increase. Neutropenia protected the vascular barrier function against acid injury. This study tests whether active oxygen species and elastase (which are presumably released by adherent PMNs) affect the microvascular barrier. Anesthetized rats underwent tracheostomy and insertion of a cannula into a lung segment. This was followed by localized instillation of 0.1 N HCl (n = 18) or saline (n = 18). Sequestration of PMNs in acid-aspirated and nonaspirated segments was 77 and 46 PMNs/high-power field (HPF), respectively, which was higher than control values of 11 and 8 PMNs/10 HPF in saline-aspirated and nonaspirated regions (P less than 0.05). Acid aspiration was associated with increased protein concentration in bronchoalveolar lavage (BAL) fluid to 3,550 and 2,900 micrograms/ml in the aspirated and nonaspirated lungs, respectively, which were higher than control values of 420 and 400 micrograms/ml (P less than 0.05). Acid aspiration also led to increased lung wet-to-dry weight ratios (W/D) of 6.6 and 5.4, which were higher than control values of 3.4 and 3.3 (P less than 0.05). Intravenous treatment of rats (n = 18) 90 min after aspiration with scavengers of reactive oxygen species, superoxide dismutase (1,500 U/kg), and catalase (5,000 U/kg), both conjugated to polyethylene glycol, did not reduce PMN sequestration but attenuated acid aspiration-induced increase in protein accumulation in BAL fluid in the aspirated and nonaspirated segments (990 and 610 micrograms/ml) as well as the increased lung W/D (4.6 and 4.0; all P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)