Anthony J. Spano

Molecular cloning, nuclear gene structure, and developmental expression of NADPH: protochlorophyllide oxidoreductase in pea (Pisum sativum L.)

Complementary DNA clones and a corresponding nuclear gene (lpcr) encoding the NADPH-dependent protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) have been characterized from pea (Pisum sativum L.). The pea lpcr gene encodes a 43 118 Da precursor polypeptide comprised of a transit peptide of 64 amino acids and a mature protein of 336 amino acids. The coding portion of the gene is interrupted by four introns, two of which are located within the transit peptide coding portion of the gene. The deduced primary structure for the pea protein is similar to those reported for Arabidopsis and two monocot species. Northern blot analysis revealed little to no decrease in steady-state levels of mRNA encoding the enzyme in etiolated leaves illuminated with continuous white light for up to 48 h. In contrast, western blot analysis showed that the major immunoreactive species present in whole leaf extracts decreased to nearly undetectable levels during this same 48 h period. These results suggest that pchlide reductase activity in pea is primarily regulated post-transcriptionally, most likely at the level of translation initiation/elongation or protein turnover.

Aberrant Localization of the Neuronal Class III b-Tubulin in Astrocytomas A Marker for Anaplastic Potential

c Background.—The class III b-tubulin isotype (bIII) is widely regarded as a neuronal marker in development and neoplasia. In previous work, we have shown that the expression of bIII in neuronal/neuroblastic tumors is differentiation dependent. In contrast, the aberrant localization of this isotype in certain nonneuronal neoplasms, such as epithelial neuroendocrine lung tumors, is associated with anaplastic potential. Objective.—To test the generality of this observation, we investigated the immunoreactivity profile of bIII in astrocytomas. Design.—Sixty archival, surgically excised astrocytomas (8 pilocytic astrocytomas, WHO grade 1; 18 diffuse fibrillary astrocytomas, WHO grade 2; 4 anaplastic astrocytomas, WHO grade 3; and 30 glioblastomas, WHO grade 4), were studied by immunohistochemistry using anti-bIII monoclonal (TuJ1) and polyclonal antibodies. A monoclonal antibody to Ki-67 nuclear antigen (NC-MM1) was used as a marker for cell proliferation. Antibodies to glial fibrillary acidic protein (GFAP) and BM89 synaptic vesicle antigen/synaptophysin were used as glial and neuronal markers, respectively. Results.—The bIII immunoreactivity was significantly greater in high-grade astrocytomas (anaplastic astrocytomas and glioblastomas; median labeling index [MLI], 35%; interquartile range [IQR], 20%‐47%) as compared with diffuse fibrillary astrocytomas (MLI, 4%; IQR, 0.2%‐21%) (P , .0001) and was rarely detectable in pilocytic astrocytomas (MLI, 0%; IQR, 0%‐0.5%) (P , .0001 vs high-grade astrocytomas; P , .01 vs diffuse fibrillary astrocytomas). A highly significant, grade-dependent relationship was observed between bIII and Ki-67 labeling and malignancy, but this association was stronger for Ki-67 than for bIII (bIII, P , .006; Ki-67, P , .0001). There was co-localization of bIII and GFAP in neoplastic astrocytes, but no BM89 synaptic vesicle antigen/synaptophysin staining was detected. Conclusions.—In the context of astrocytic gliomas, bIII immunoreactivity is associated with an ascending gradient of malignancy and thus may be a useful ancillary diagnostic marker. However, the significance of bIII-positive phenotypes in diffuse fibrillary astrocytomas with respect to prognostic and predictive value requires further evaluation. Under certain neoplastic conditions, bIII expression is not neuron specific, calling for a cautious interpretation of bIII-positive phenotypes in brain tumors. (Arch Pathol Lab Med. 2001;125:613‐624)

Genetically targeted magnetic control of the nervous system

Optogenetic and chemogenetic actuators are critical for deconstructing the neural correlates of behavior. However, these tools have several limitations, including invasive modes of stimulation or slow on/off kinetics. We have overcome these disadvantages by synthesizing a single-component, magnetically sensitive actuator, “Magneto,” comprising the cation channel TRPV4 fused to the paramagnetic protein ferritin. We validated noninvasive magnetic control over neuronal activity by demonstrating remote stimulation of cells using in vitro calcium imaging assays, electrophysiological recordings in brain slices, in vivo electrophysiological recordings in the brains of freely moving mice, and behavioral outputs in zebrafish and mice. As proof of concept, we used Magneto to delineate a causal role of striatal dopamine receptor 1 neurons in mediating reward behavior in mice. Together our results present Magneto as an actuator capable of remotely controlling circuits associated with complex animal behaviors.

Differential distribution of the neuron-associated class III β-tubulin in neuroendocrine lung tumors

OBJECTIVE To study the immunoreactivity profile of the neuron-associated class III beta-tubulin isotype (beta III) in epithelial lung tumors. DESIGN One hundred four formalin-fixed, paraffin-embedded primary and metastatic lung cancer specimens were immunostained with an anti-beta III mouse monoclonal antibody (TuJ1) and an anti-beta III affinity-purified rabbit antiserum. Paraffin sections from fetal, infantile, and adult nonneoplastic lung tissues were also examined. RESULTS In the fetal airway epithelium, beta III staining is detected transiently in rare Kulchitsky-like cells from lung tissues corresponding to the pseudoglandular and canalicular but not the saccular or alveolar stages of development. beta III is absent in healthy, hyperplastic, metaplastic, and dysplastic airway epithelium of the adult lung. In contrast, beta III is highly expressed in small cell lung cancer, large cell neuroendocrine carcinoma, and in some non-small cell lung cancers, particularly adenocarcinomas. There is no correlation between expression of beta III and generic neuroendocrine markers, such as chromogranin A and/or synaptophysin, in pulmonary adenocarcinomas. Also, focal beta III staining is present in primary and metastatic adenocarcinomas (to the lung) originating in the colon, prostate, and ovary. beta III is expressed to a much lesser extent in atypical carcinoids and is rarely detectable in typical carcinoids and squamous cell carcinomas of the lung. The distribution of beta III in small cell lung cancer and adenocarcinoma metastases to regional lymph nodes and brain approaches 100% of tumor cells, which is substantially greater than in the primary tumors. CONCLUSIONS In the context of neuroendocrine lung tumors, beta III immunoreactivity is a molecular signature of high-grade malignant neoplasms (small cell lung cancer and large cell neuroendocrine carcinoma) although its importance in atypical carcinoids must be evaluated further. In addition, beta III may be a useful diagnostic marker in distinguishing between small cell lung cancers and certain non-small cell lung cancers (poorly differentiated squamous cell carcinomas), especially in small biopsy specimens. To our knowledge, beta III is the only tumor biomarker that exhibits a substantially more widespread distribution in poorly differentiated than in better differentiated pulmonary neuroendocrine tumors. However, the significance of beta III phenotypes in non-small cell lung cancer, particularly adenocarcinoma, with respect to neuroendocrine differentiation and prognostic value, requires further evaluation.

Expression of stathmin family genes in human tissues: non-neural-restricted expression for SCLIP.

The stathmin family consists of phosphoproteins highly conserved in vertebrates and thought to be implicated in the development and functional regulation of various organs, most notably the nervous system. This family includes stathmin, SCG10, SCLIP, and RB3, phosphoproteins that are related by structural and functional homologies. They all sequester tubulin and interfere with microtubule dynamics, a property due to their shared stathmin-like domain. Little is known about the expression of the stathmin gene family in humans. Herein, we describe for the first time, for a collection of human tissues, the expression of each member of this family, using real-time quantitative RT-PCR. We found that stathmin is ubiquitously expressed, whereas SCG10 and RB3 are neural enriched, expression patterns similar to those reported for other mammals. Surprisingly, SCLIP, whose expression is thought to be neural-specific, exhibits a broader tissue distribution. Analyses of the SCLIP gene (approved symbol STMN3) show that it contains several NRSE-like elements that display low or no affinity for the cognate binding protein NRSF. The substantial expression of SCLIP in most tissues points out a novel function for this protein outside the nervous system and raises the possibility that its coexpression with stathmin could provide some degree of functional redundancy.

Increasing Efficiency of Photoelectronic Conversion by Encapsulation of Photosynthetic Reaction Center Proteins in Arrayed Carbon Nanotube Electrode

The construction of efficient light energy converting (photovoltaic and photoelectronic) devices is a current and great challenge in science and technology and one that will have important economic consequences. Here we show that the efficiency of these devices can be improved by the utilization of a new type of nano-organized material having photosynthetic reaction center proteins encapsulated inside carbon nanotube arrayed electrodes. In this work, a generically engineered bacterial photosynthetic reaction center protein with specifically synthesized organic molecular linkers were encapsulated inside carbon nanotubes and bound to the inner tube walls in unidirectional orientation. The results show that the photosynthetic proteins encapsulated inside carbon nanotubes are photochemically active and exhibit considerable improvement in the rate of electron transfer and the photocurrent density compared to the material constructed from the same components in traditional lamella configuration.

Class III β-Tubulin isotype (β III) in the adrenal medulla: I. Localization in the developing human adrenal medulla

The class III β‐tubulin isotype (β III) is present in neurons of the central and peripheral nervous systems at the earliest stages of morphological differentiation (Easter et al., J Neurosci 13:285–299, 1993; Katsetos et al., J Neuropathol Exp Neurol 52:655–666, 1993). The localization of this protein by immunohistochemistry in the different cell types of the developing human adrenal medulla is described.

Structure and Expression of Chloroplast-Localized Porphobilinogen Deaminase from Pea (Pisum sativum L.) Isolated by Redundant Polymerase Chain Reaction

Porphobilinogen (PBG) deaminase catalyzes the polymerization of four PBG monopyrrole units into the linear tetrapyrrole hydroxymethylbilane necessary for the formation of chlorophyll and heme in plant cells. Degenerate oligonucleotide primers were designed based on amino acid sequence data (generated by mass spectrometry) for purified PBG deaminase from pea (Pisum sativum L.) chloroplasts. These primers were used in Taql polymerase-catalyzed polymerase chain reaction (PCR) amplification to produce partial cDNA and nuclear genomic fragments encoding the enzyme. Subsequently, a 1.6-kb cDNA was isolated by screening a cDNA library constructed in [lambda]gt11 from leaf poly(A)+ RNA with the PCR products. The cDNA encodes an approximately 40-kD polypeptide containing a 46-amino acid NH2-terminal transit peptide and a mature protein of 323 amino acids. The deduced amino acid sequence of the mature pea enzyme is similar to PBG deaminases from other species and contains the conserved arginine and cysteine residues previously implicated in catalysis. Northern blot analysis indicates that the pea gene encoding PBG deaminase is expressed to varying levels in chlorophyll-containing tissues and is subject to light induction.

β-tubulin isotype classes II and V expression patterns in nonsmall cell lung carcinomas

Previous studies suggest that beta-tubulin isotype protein levels could be useful as indicators of nonsmall cell lung cancer (NSCLC) aggressiveness. However, measurement of protein amounts in tissue samples by staining techniques is semiquantitative at best. Since technologies for measuring mRNA levels have become more efficient and quantitative, we wanted to determine whether beta-tubulin message levels may be useful as biomarkers. Quantitative real-time RT-PCR was used to measure the seven classes of beta-tubulin isotypes, stathmin and MAP4 mRNA levels in 64 NSCLC and 12 normal lung tissue samples. We found significantly higher fractions of beta-tubulin classes II and V mRNA compared to the other isotypes in all lung tumor samples (P < 0.05). In addition, the ratio of beta-tubulin classes II/V mRNA was significantly higher in NSCLCs compared to normal lung tissues (P < 0.001). The data suggest that the ratio of beta-tubulin classes II and V mRNA could be useful as a biomarker for NSCLC tumor differentiation and/or NSCLC aggressiveness. Furthermore, the ratio of MAP4 to stathmin mRNA was found to be higher in diseased lung tissues compared to normal lung tissues, suggesting this ratio might also be used as a clinically relevant biomarker for NSCLCs.

Isolation, characterization and partial amino acid sequence of a chloroplast-localized porphobilinogen deaminase from pea (Pisum sativum L.)

Porphobilinogen deaminase catalyzes the condensation of four porphobilinogen monopyrrole units into hydroxymethylbilane, a linear tetrapyrrole necessary for the formation of chlorophyll and heme in higher plant cells. We report the purification to homogeneity of a chloroplast-localized form of the enzyme from pea (Pisum sativum L.) by a novel purification scheme involving dye-ligand affinity chromatography. The purified chloroplast porphobilinogen deaminase consists of a single polypeptide with a relative molecular mass of 36-45 kDa as determined by size-exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of the protein is acidic. The activity of the enzyme shows different levels of sensitivity to divalent cations and is most sensitive to FE2+. The amino terminus of pea enzyme has been obtained by microsequencing and determined to bear little similarity to the amino acid sequences of porphobilinogen deaminases purified from other organisms. Polyclonal antisera elicited against the purified protein has been used to examine the abundance and cellular distribution of the enzyme.