Comparative study of bovine ocular lens proteins in native tissue and an extract using the x-ray diffraction method

It has been shown that the maxima (Bragg-spacings 4,5-19 A) on the X-ray diffraction patterns of the bovine lens native tissues from nuclear and cortical parts are predominantly due to the water-soluble crystallin intramolecular structure. The structures of water-soluble and water-insoluble fractions from bovine lens nucleus and cortex were qualitatively compared. Reversible dependence of the lens water-soluble protein structure on water content in the system was demonstrated.     

Target tissue specificity of retinoic acid-induced stress proteins and malformations in mice

Retinoic acid (RA) is teratogenic in rodents and also induces the synthesis of stress proteins in fetal mouse limb buds. To determine if the RA induction of stress proteins is target tissue specific, pregnant CD-1 mice were gavaged with 100 mg/kg RA on day 11 of gestation, and nuclei isolated from tissues susceptible to RA-induced malformations (target tissues) as well as nuclei isolated from nontarget tissues were examined for stress protein synthesis and malformations. Forelimb and hindlimb (target tissues), as well as heart and tail (nontarget tissues), were removed from embryos 2.5 hours after RA treatment (1.5 hr after [3H]leucine labeling). Cell nuclei were isolated, stained with a DNA specific fluorochrome, propidium iodide, and sorted from the G0 + G1 and G2 + M phases of the cell cycle. Forelimb and hindlimb target tissues showed the synthesis in these embryonic nuclear proteins of an 84,000 relative molecular mass (Mr) protein and a 90,000 Mr protein following RA treatment. Two 20,000-25,000 Mr stress proteins were also labeled both in forelimb and hindlimb. Forelimb and hindlimb from untreated dams showed no stress protein labeling. Neither heart nor tail, nontarget tissues, showed any stress protein labeling following RA treatment. Classical teratological evaluation of embryos treated on GD 11 and sacrificed on GD 17 showed that 100% of the fetuses had forelimb and/or hindlimb malformations, while no malformations were observed in either the heart or tail. Based on the correlation of teratological anomalies with the identification of stress proteins in target tissue only, we postulate that stress proteins may be involved in the teratogenic process. Further work is necessary to establish whether a causal relationship exists.     

Isolation and Properties of Nuclei from Control and Auxin-treated Soybean Hypocotyl

A quick procedure for the isolation of nuclei with good yield from soybean hypocotyl (Glycine max var. Wayne) was developed. The isolated nuclei appeared to retain their structural integrity. They were typically ellipsoidal with minima and maxima diameter of about 6 and 8 to 10 micrometers. While the nuclei were similar in size, the nucleoli were significantly larger in nuclei from auxin-treated tissue. The DNA content per nucleus was 4 ± 1 picograms for both untreated and auxin-treated tissues. The DNA: RNA: protein ratio of isolated nuclei in untreated and auxin-treated tissues was 1: 3.1: 11 and 1: 5.4: 21.7, respectively. The purified nuclei were active in RNA synthesis; the level of RNA polymerase II activity expressed in the nuclei from untreated tissue was 50 to 60% higher than RNA polymerase. I. The nuclei from auxin-treated tissues contained about 2.5 times as much RNA polymerase I activity as nuclei from untreated tissue. The purified nuclei from both untreated and auxin-treated tissues were also active in the incorporation of ³H-TTP into DNA.     

Early tissue interactions leading to embryonic lens formation in Xenopus laevis

Our previous research has demonstrated that lens induction in Xenopus laevis requires inductive interactions prior to contact with the optic vesicle, which classically had been thought to be the major lens inductor. The importance of these early interactions has been verified by demonstrating that lens ectoderm is specified by the time it comes into contact with the optic vesicle. It has been argued that the tissues which underlie the presumptive lens ectoderm during gastrulation and neurulation, dorsolateral endoderm and mesoderm, are the primary early inductors. We show here, however, that these tissues alone cannot elicit lens formation in Xenopus ectoderm. Evidence is presented that presumptive anterior neural plate tissue (which includes the early eye rudiment) is an essential early lens inductor in Xenopus. The presence of dorsolateral mesoderm appears to enhance this response. These findings support a model in which an essential inductive signal passes through the plane of ectoderm during gastrula and early neurula stages from presumptive anterior neural tissue to the presumptive lens ectoderm. Since there is evidence for such interactions within a tissue layer in mesodermal and neural induction as well, this may be a general feature of the initial stages of determination of many tissues.     

Matricellular protein SPARC is translocated to the nuclei of immortalized murine lens epithelial cells

The matricellular glycoprotein, secreted protein acidic and rich in cysteine (SPARC), has complex biological activities and is important for lens epithelial cell function and regulation of cataract formation. To understand how SPARC influences lens epithelial cell activity and homeostasis, we have studied the subcellular distribution of SPARC in murine lens epithelial cells in vitro. We demonstrate that endogenous SPARC is located in the cytoplasm of either quiescent or dividing lens epithelial cells in culture. However, cytoplasmic SPARC was translocated into the nuclei of immortalized lens epithelial cells upon a significant reduction of intracellular SPARC in these cells. Recombinant human (rh) SPARC added to the culture media was quickly and efficiently internalized into the cytosol of SPARC-null lens epithelial cells. Moreover, cytoplasmic rhSPARC was also translocated into the nucleus after exogenous rhSPARC was removed from the culture media. The translocation of SPARC into the nucleus was therefore triggered by the reduction of SPARC protein normally available to the cells. A mouse SPARC-EGFP chimeric fusion protein (70 kDa) was expressed in lens epithelial cells and 293-EBNA cells, and was observed both in the cytoplasm and culture medium, but not in the nucleus. SPARC does not appear to have a strong nuclear localization sequence. Alternatively, SPARC might pass through the nuclear pore complex by passive diffusion. SPARC therefore functions not only as an extracellular protein but also potentially as an intracellular protein to influence cellular activities and homeostasis.     

Perturbing the ubiquitin pathway reveals how mitosis is hijacked to denucleate and regulate cell proliferation and differentiation in vivo

Background

The eye lens presents a unique opportunity to explore roles for specific molecules in cell proliferation, differentiation and development because cells remain in place throughout life and, like red blood cells and keratinocytes, they go through the most extreme differentiation, including removal of nuclei and cessation of protein synthesis. Ubiquitination controls many critical cellular processes, most of which require specific lysines on ubiquitin (Ub). Of the 7 lysines (K) least is known about effects of modification of K6.

Methodology and Principal Findings

We replaced K6 with tryptophan (W) because K6 is the most readily modified K and W is the most structurally similar residue to biotin. The backbone of K6W-Ub is indistinguishable from that of Wt-Ub. K6W-Ub is effectively conjugated and deconjugated but the conjugates are not degraded via the ubiquitin proteasome pathways (UPP). Expression of K6W-ubiquitin in the lens and lens cells results in accumulation of intracellular aggregates and also slows cell proliferation and the differentiation program, including expression of lens specific proteins, differentiation of epithelial cells into fibers, achieving proper fiber cell morphology, and removal of nuclei. The latter is critical for transparency, but the mechanism by which cell nuclei are removed has remained an age old enigma. This was also solved by expressing K6W-Ub. p27^kip, a UPP substrate accumulates in lenses which express K6W-Ub. This precludes phosphorylation of nuclear lamin by the mitotic kinase, a prerequisite for disassembly of the nuclear membrane. Thus the nucleus remains intact and DNAseIIβ neither gains entry to the nucleus nor degrades the DNA. These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues.

Conclusions and Significance

K6W-Ub provides a novel, genetic means to study functions of the UPP because it can be targeted to specific cells and tissues. A fully functional UPP is required to execute most stages of lens differentiation, specifically removal of cell nuclei. In the absence of a functional UPP, small aggregate prone, cataractous lenses are formed.     

Fast quantification of immunohistochemistry tissue microarrays in lung carcinoma

Tissue microarrays (TMAs) are an effective tool for high-throughput molecular analysis of tissues to help identify new diagnostic and prognostic markers and targets in human cancers. We have developed a fully automated method for rapid, continuous and quantitative analysis of TMAs based on immunohistochemistry. The method deals with complex and varying tissue architectures, segments tumour cells from normal cells, conducts cell compartmentalisation, identifies nuclei and cytoplasm and produces three different continuous measurements of marker expression levels within tumour cell nuclei, tumour cell cytoplasm and total tumour cell protein expression. We have demonstrated this method using three independent protein markers (BAK, BAX and a novel biomarker, named KS) over 7 TMAs, involving 2 BAK stained TMAs with 229 tumour tissue cores, 2 BAX stained TMAs with 229 tumour tissue cores and 3 KS stained TMAs with 373 tumour cores of lung carcinomas. We validated the automated method, showing that the automated scoring is significantly correlated with the pathologist-based scoring.     

Subcellular localization of viroids in highly purified nuclei from tomato leaf tissue

Approximately 95% of the viroid RNA which is present in potato spindle tuber viroid (PSTV)-infected tomato plant leaf issue, is associated with the nucleolar fraction obtained from purified nuclei. Viroids were released from the nucleolar fraction by increasing the ionic strength of the medium to 0.66 suggesting that viroid RNA is present in these subnuclear components in a protein-nucleic acid complex. A purification procedure for nuclei from leaf tissue had to be newly developed; it involves two Percoll density centrifugations as final steps. The nuclei were sonicated and the sonicate fractionated into fractions either highly enriched in nucleoli or in broken chromatin and ribonucleoprotein particles. The viroid content in the different samples was determined by gel electrophoresis. Depending upon the progress of the disease, viroid copy numbers between 200 and 10,000 per cell were observed in homogenized tissue, purified nuclei and in the nucleolar fraction. In chloroplasts, practically no viroids were detected. The results are discussed in the light of current hypotheses about the replication, pathogenicity and origin of viroids.     

Quantitation and characterization of thyrotropin-releasing hormone in vagal nuclei and other regions of the medulla oblongata of the rat

Abstract: Since evidence is now available to support a nonendocrine autonomic function for thyrotropin-releasing hormone (TRH), quantitative measurements of TRH were made in nuclei of the vagal complex and other areas of the caudal medulla oblongata of the rat. Regions containing the dorsal motor nucleus of the vagus (DMN), nucleus tractus solitarius (NTS), hypoglossal nucleus, dorsal column nuclei, descending nucleus V (DNV), nucleus ambiguus (NA), raphe nuclei (MR) dorsomedial and ventromedial reticular formation, and inferior olivary nuclei were isolated from 300-μm-thick frozen sections of medulla by the micropunch technique. Each region was pooled bilaterally, homogenized in 0.1 M HCl, and vacuum-dried. Extracts were assayed for TRH by specific radioimmunoassay (RIA). TRH levels varied 100-fold among medulla nuclei. Highest content (ng/mg protein ± SEM) was found in DMN (14 ± 1.38) and NTS (4.7 ± 0.68), whereas lowest levels occurred in the DNV and MR (0.13, 0.06). Nearly 65% of the total medullary TRH was localized in nuclei associated with vagal complex (DMN, NTS, NA). Characterization of tissue immunoreactivity (TRHi) in these regions suggests the presence of TRH, since (1) medullary tissue extracts competed with ¹²⁵I-TRH for antibody binding sites with the same affinity as authentic TRH; (2) TRHi in tissue extracts co-migrated with synthetic TRH when subjected to reverse-phase high performance liquid chromatography and Sephadex G-10 chromatography; and (3) rat serum TRH peptidases degraded TRHi and authentic TRH at similar rates. Another group of rats was subjected to unilateral (right side) vagotomy. At 33 weeks post-vagotomy, the vagal preganglionic cell population in the ipsilateral DMN was depleted 50–75%, while the contralateral side was unaffected. Interestingly, the content of TRH in the ipsilateral (right) DMN remained unchanged, whereas TRH in the contralateral DMN increased by 50%. In contrast, TRH was significantly elevated in the NA on the ipsilateral side of the lesion. TRH in both ipsi- and contralateral NTS was unchanged when compared with sham-operated controls. These results indicate that (1) TRH is present in several specific loci of the medulla; (2) very high levels are found in the vagal complex; and (3) vagotomy may alter TRH in the contralateral DMN and ipsilateral NA.     

The apparent absence of lamin B1 and emerin in many tissue nuclei is due to epitope masking

Summary Immunolocalization studies have concluded that the nuclear membrane protein, emerin, is absent from many cell types and that lamin B1 is absent from adult heart and skeletal muscle. We now show that epitope masking in the nucleus is often responsible for failure to detect emerin and lamins in human, rat and pig tissues. Human heart cardiomyocyte nuclei were negative for lamin B1 using a commercial mAb, but were positive using two other lamin B1 antibodies, mAb8D1 and pAbB1-cbs. Rat hippocampal neuronal nuclei were immunostained by mAb8D1, but not pAbB1-cbs, while the commercial antibody stained only a subset. These data suggest that different regions of the lamin B1 molecule are masked in different tissues. Similarly, pig spleen had fewer emerin-positive nuclei than lung (5% vs. 32%), although their emerin content was similar by Western blotting. As mAbs against six epitopes gave the same result, the whole emerin molecule is either masked or redistributed in a subset of cells. Our findings argue that immunostaining evidence can be misleading for expression of nuclear envelope proteins. Problems with lamin B1 immunostaining can be avoided by using mAb8D1, but use of antibodies recognizing different epitopes may reveal cell-specific protein interactions in the nucleus.     

Adipose tissue of Drosophila melanogaster: VII. Distribution of nuclear DNA amounts along the anterior-posterior axis in the larval fat body

The DNA content of fat body nuclei was measured cytophotometrically as a function of position along an anterior-posterior (A-P) axis of the tissue throughout larval development. There was a dramatic 55-fold increase in the average amount of DNA per nucleus during the first 3 days of this period, but there was no further increase during the final day. However, the rate of increase had regional specificity. The amount of DNA per nucleus correlated significantly with its position along the A-P axis of the tissue: nuclei in a posterior direction contain gradually increasing amounts of DNA. There was up to a seven-fold difference between the smallest anterior and largest posterior nucleus. In addition, for three of the ages studied there was a subgradient in the posterior region with a slope that was considerably steeper than that of the overall tissue gradient. The tissue has three characteristic morphological regions, anterior, medial, and posterior, which can be recognized early in development and which are maintained throughout the larval period. The distribution of nuclear DNA classes determined for cells in each region for the final 2 days of larval life became fixed before the final day of development. The significance of the DNA gradient in terms of a protein storage gradient is discussed.     

Immunoproteasome expression in a nonimmune tissue,the ocular lens

Interferon gamma (IFN gamma) induces the expression of three catalytic subunits of the 20S proteasome that can replace their constitutive homologues to form the "immunoproteasome," named to reflect its antigen presentation function. However, immunoproteasome levels and their modulation in nonimmune tissues remain unknown. A disrupted lens differentiation program observed in transgenic mice that constitutively express IFN gamma in the immune-privileged lens tissue suggests a role for this cytokine in differentiation. We have developed a competitive RT-PCR assay that demonstrates substantially increased levels of immuno subunits and unchanged levels of constitutive subunits in transgenic compared to wild-type lenses. Similar results were observed with IFN gamma treated alpha TN4-1 lens epithelial cells. A comparison of these subunits in different immune and nonimmune mouse tissues revealed unique expression patterns. The presence of immuno subunits in nonimmune tissues such as lens suggests that the immunoproteasome may also have nonimmune functions, such as that in lens differentiation.     

Localization of arginine decarboxylase in tobacco plants

The lack of knowledge about the tissue and subcellular distribution of polyamines (PAs) and the enzymes involved in their metabolism remains one of the main obstacles in our understanding of the biological role of PAs in plants. Arginine decarboxylase (ADC; EC 4.1.1.9) is a key enzyme in polyamine biosynthesis in plants. We have characterized a cDNA coding for ADC from Nicotiana tabacum L. cv. Petit Havana SR1. The deduced ADC polypeptide had 721 amino acids and a molecular mass of 77 kDa. The ADC cDNA was overexpressed in Escherichia coli , and the ADC fusion protein obtained was used to produce polyclonal antibodies. Using immunological methods, we demonstrate the presence of the ADC protein in all plant organs analysed: flowers, seeds, stems, leaves and roots. Moreover, depending on the tissue, the protein is localized in two different subcellular compartments, the nucleus and the chloroplast. In photosynthetic tissues, ADC is located mainly in chloroplasts, whereas in non-photosynthetic tissues the protein appears to be located in nuclei. The different compartmentation of ADC may be related to distinct functions of the protein in different cell types.