Lung nodules in a silver polisher.
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Adult↗
Biomedical subjects
S P Bhat
Publications and source records attributed to S P Bhat.
The ocular lens epithelium.
An adult lens contains two easily discernible, morphologically distinct compartments, the epithelium and the fiber-cell mass. The fiber-cell mass provides the lens with its functional phenotype, transparency. Metabolically, in comparison to the fiber cells the epithelium is the more active compartment of the ocular lens. For the purposes of this review we will only discuss the surface epithelium that covers the anterior face of the adult ocular lens. This single layer of cells, in addition to acting as a metabolic engine that sustains the physiological health of this tissue, also works as a source of stem cells, providing precursor cells, which through molecular and morphological differentiation give rise to fiber cells. Morphological simplicity, defined developmental history and easy access to the experimenter make this epithelium a choice starting material for investigations that seek to address universal questions of cell growth, development, epithelial function, cancer and aging. There are two important aspects of the lens epithelium that make it highly relevant to the modern biologist. Firstly, there are no known clinically recognizable cancers of the ocular lens. Considering that most of the known malignancies are epithelial in origin this observation is more than an academic curiosity. The lack of vasculature in the lens may explain the absence of tumors in this tissue, but this provides only a teleological basis to a very important question for which the answers must reside in the molecular make-up and physiology of the lens epithelial cells. Secondly, lens epithelium as a morphological entity in the human lens is first recognizable in the 5th-6th week of gestation. It stays in this morphological state as the anterior epithelium of the lens for the rest of the life, making it an attractive paradigm for the study of the effects of aging on epithelial function. What follows is a brief overview of the present status and lacunae in our understanding of the biology of the lens epithelium.
Adult↗
Canonical heat shock element in the alpha B-crystallin gene shows tissue-specific and developmentally controlled interactions with heat shock factor.
Oligomerization of the heat shock factor (HSF) and its interaction with the heat shock element (HSE) are the hallmark of active transcriptional response to tangible physical or chemical stress. It is unknown if these interactions are subject to control and modulation by developmental cues and thus have tissue or stage specificity. By using promoter sequences containing a canonical HSE from the alphaB-crystallin gene, we demonstrate a tissue-specific transition from monomeric (in fetal and early neonatal stages that lack oligomeric HSF.HSE complexes) to oligomeric HSF-HSE interactions by postnatal day 10-21 in the ocular lens. Developmental control of these interactions is further demonstrated by induction of oligomeric HSF.HSE complexes in neonatal extracts by in vitro manipulations, interestingly, only in the lens and not in the brain, heart, or liver extracts. The exclusive presence of oligomeric HSF.HSE complexes in the postnatal/adult lens corresponds to known highly increased number of alphaB-crystallin transcripts in this tissue.
Animals↗
Ectopic expression of alpha B-crystallin in Chinese hamster ovary cells suggests a nuclear role for this protein.
alpha B-crystallin (alpha B) is known to be a cytosolic, small heat shock-like multimeric protein that has anti-aggregation, chaperone-like properties. The expression of the alpha B-crystallin gene is developmentally regulated and is induced by a variety of stress stimuli. Importantly, alpha B-crystallin expression is enhanced during oncogenic transformation of cells, in a number of tumors, and most notably, in many neurodegenerative disorders, including Alzheimer's disease and multiple sclerosis. Other than its perceived role as a structural protein in the ocular lens, the actual function of alpha B-crystallin in cellular physiology remains unknown. We have stably transfected CHO cells with an inducible alpha B-cDNA-MMTV-promoter construct that allows the synthesis of recombinant alpha B-crystallin only upon exposure of these cells to dexamethasone. Using immunostaining and conventional and confocal microscopy, we have examined the subcellular distribution of the ectopically expressed alpha B-crystallin. We find that in addition to being in the cytoplasm, the protein resides in the nuclear interior in the interphase nucleus. Double labeling with anti alpha B-crystallin and anti-tubulin, concanavallin, and wheat germ agglutinin, respectively, revealed that during cell division alpha B-crystallin is excluded from condensed chromatin and the nascent nuclei. However, the protein again appears in the newly formed nuclei after the completion of cytokinesis suggesting a conditional, regulatory role for alpha B-crystallin in the nucleus.
Animals↗
Quantitative estimation of RNA transcripts suggests persistence of Pax-6 expression in the postembryonic chick retina.
Pax-6 is expressed during early embryonic development of the eye. Very little is known about its expression in the functionally mature retina. We have detected Pax-6 transcripts in the ganglion cell- and amacrine cell layers at days 3, 10, 17 and 31 posthatching and in 2- to 3-month-old chick retina by in situ hybridization. These observations were confirmed by a quantitative analysis. Competitive RT-PCR with a homologous internal control revealed a significant reduction (p < 0.001) in the number of Pax-6 transcripts in day 17 retina [(0.39 +/- 0.13) x 10(10)/mg tissue] compared to day 3 retina [(1.65 +/- 0.48) x 10(10)/mg tissue]. Although significantly lower than at day 3, the day 31 retina [(0.7 +/- 0.16) x 10(10)/mg tissue] and retina from 2- to 3-month-old chicks [(0.9 +/- 0.28) x 10(10)/mg tissue] contained an increased number of Pax-6 transcripts in comparison to day 17. On the basis of the amount of RNA, the number of Pax-6 transcripts in the day 3 retina [(0.45 +/- 0.14) x 10(10)/microg RNA] relative to day 17 retina [(0.4 +/- 0.08) x 10(10)/microg RNA] did not change significantly (p = 0.29). However, at day 31 and at 2-3 months of age an increased number of Pax-6 transcripts [(0.65 +/- 0.14) x 10(10)/microg RNA and (0.65 +/- 0.2) x 10(10)/microg RNA, respectively)] were found. In view of the known association of Pax-6 expression with proliferation and emergence of different cell types, these data suggest that cell types in ganglion and inner nuclear cell layers may retain proliferative potential for an extended period in the young adult retina.
Aging↗
Expression and synthesis of the Na,K-ATPase beta 2 subunit in human retinal pigment epithelium.
Na,K-ATPase in the retinal pigment epithelium (RPE) is apically localized, whereas in most other tissues this pump is found predominantly in the basolateral membrane domain. As part of our investigations into the molecular aspects of this pump in the RPE, we have cloned the cDNA and characterized the expression of the gene encoding the beta 2 subunit isoform of Na,K-ATPase in human, rat and bovine RPE and in the bovine choroid plexus. We have also detected the beta 2 isoform polypeptide in the human RPE (hRPE). Comparison of complete coding sequences derived from cloned cDNAs revealed that all beta 2 sequences from RPE, and the choroid plexus, differed uniformly at positions: P51/L, M121/I, and L148/R from the published sequences for human retina and liver. However, analysis of 10 RT-PCR clones derived from 5 fetal and 2 adult human retinas sequenced in our laboratory, revealed that only the P51/L residue was different with the hRPE beta 2 subunit sequence. Northern blot analysis indicated a 3.4-kb RNA transcript for the beta 2 subunit, a 4.5-kb RNA for the alpha 1 subunit and a doublet of 2.3 and 2.6 kb for the beta 1 subunit, respectively. alpha 1 (100 kDa), beta 1 (45 kDa) and beta 2 (65 kDa) isoforms were detected in hRPE extracts by immunoblotting. No alpha 2 and alpha 3 RNA transcripts were found in the hRPE. Quantification of beta 2 mRNA by RT-PCR revealed 2.7 x 10(5) molecules per ng of poly A+ RNA. This is similar to the beta 1 isoform levels reported previously from our laboratory. These data demonstrate the coexistence of significant amounts of alpha 1, beta 1 and beta 2 Na,K-ATPase subunits in the RPE. It is therefore reasonable to suggest that both alpha 1 beta 1 and alpha 1 beta 2 heterodimers are present in these cells.
Adult↗
Expression of recombinant alpha Ains-crystallin and not alpha A-crystallin inhibits bacterial growth.
alpha A-Crystallin and alpha Ains-crystallin are derived from the alpha A-crystallin gene via alternative splicing. They are identical except for the presence of a polypeptide, 23 amino acids long, encoded by the 'insert' exon. Evolutionary logic would suggest that the insertion of a 23 amino acid peptide in the middle of alpha A-crystallin, a protein evolving more slowly than either histone H1, cytochrome c or hemoglobin, would lead to appreciable structural and functional changes. However, based on physico-chemical studies, it is presently believed that alpha A-crystallin and alpha Ains-crystallin are functionally equivalent and that the presence of the 'insert' peptide in alpha Ains-crystallin is inconsequential. We report here that the independent expression of recombinant alpha Ains-crystallin, and not alpha A-crystallin, inhibits growth of the bacterial host. These observations were confirmed in coexpression experiments, wherein both the proteins were expressed in the same cell. Interestingly, growth inhibition is reversible. Importantly, the data demonstrate that it is catalytic amounts and not the gross accumulation of alpha Ains-crystallin which causes growth inhibition. Given the prior knowledge that alpha A-crystallin and alpha Ains-crystallin differ by a peptide of 23 amino acids, these data suggest that the "insert peptide' in alpha Ains-crystallin imparts properties on this protein that are different from alpha A-crystallin.
Animals↗
Characterization and quantification of full-length and truncated Na,K-ATPase alpha 1 and beta 1 RNA transcripts expressed in human retinal pigment epithelium.
We have characterized cDNA clones encoding the alpha 1 and beta 1 subunits of Na,K-ATPase produced in the human retinal pigment epithelium (hRPE). In addition to isolating clones corresponding to known sequences of Na,K-ATPase subunits, we report hitherto unknown forms of Na,K-ATPase with unique deduced amino acid (aa) sequences in their C-termini. Truncated cDNA sequences were found for both the beta 1 and alpha 1 subunits. While the beta 1 sequence is truncated by two aa residues at the C terminus, in the alpha 1 sequence 342 aa have been replaced by a unique sequence containing only 44 aa. Interestingly, this new C-terminal polypeptide shows sequence similarities to the Ca(2+)-ATPase and contains consensus sequence elements for phosphorylation and cell adhesion, suggesting expression of Na,K-ATPase subunits with unique functions. Using reverse transcription-polymerase chain reaction, RNA sequences for alpha 1, beta 1 and their corresponding truncated isoforms were quantified. 4.0 x 10(5) alpha 1 and 2.3 x 10(5) beta 1 molecules were found per ng of mRNA from hRPE. Much lower levels were detected for truncated alpha 1 and beta 1 (3.6 x 10(3) and 2.7 x 10(3) molecules/ng, respectively). These data corroborate the expression of truncated transcripts coding for unique aa sequences in hRPE, and suggest that factors other than alpha 1 and beta 1 mRNA levels regulate the equimolar accumulation of alpha and beta subunits in the plasma membrane.
Amino Acid Sequence↗
Complete structure and expression of the rat alpha B-crystallin gene.
alpha B-Crystallin, a member of the small heat shock family of proteins, is synthesized as a component of various developmental programs, in response to stress and in a number of pathological states. We have determined the complete structure of the alpha B-crystallin gene (6,806 bp encompassing 2,299 bp upstream from ATG and 859 bp at the 3' end, past the first polyadenylation signal). Comparison of the rat and the human alpha B-crystallin genes reveals significant conservation of the nucleotide sequences in almost all regions except in intron 2. The 1-kb region immediately upstream of ATG shows about 75% overall homology. A 78-bp sequence in the intron 1 and sequences in the 3' untranslated region show about 95% and 85% sequence identity, respectively. Characterization of the expression of this gene in different tissues in the rat by extensive analyses, utilizing primer extension. RNase protection, and rapid amplification of cDNA ends (RACE) revealed a predominant transcription initiation site 44 bp upstream of ATG. Northern analyses with "coding-only" and upstream "noncoding" probes did not support the thesis that heterogeneity in the alpha B-crystallin mRNAs arises from variations in the sequences immediately upstream of the predominant transcription initiation site. Importantly, the known relative levels of alpha B-crystallin protein in different tissues correlate best with the presence of transcripts starting from this initiation site.
Amino Acid Sequence↗
Investigations into size heterogeneity of the alpha B-crystallin mRNA.
alpha B-crystallin is expressed in a variety of developmental, physiological and pathological conditions in a number of tissues. Based on northern blots of total RNA, the existence of at least two size classes of alpha B-crystallin mRNAs has been reported, one smaller predominant species, 0.8-0.9 kb, and the other 1.2 to 1.4 kb. This heterogeneity has been attributed to alternative upstream transcriptional initiation. We have investigated the origin of the size heterogeneity of alpha B-crystallin mRNA by using 5'-upstream-, coding- and 3'-untranslated-region probes in RNAse protection and northern blot assays. RNAse protection assays indicate that there is only one predominant initiation site as previously reported and that the second polyadenylation signal is not used in the rat gene. Importantly, northern blot data obtained with coding region-only probe shows that the size of alpha B mRNA detected in the heart and the lens is similar (0.78 kb) in poly A+ as well as in total RNA. On the other hand, in the brain and in the lung, the larger hybridizing species (1.05 kb and 1.22 kb respectively) seen in total RNA are not detected in poly A+RNA which shows a 0.95 kb species in both tissues. The 5' upstream probe (-1 to -499) produces weak hybridization patterns in the brain and the lung, similar to those obtained with coding region-only probe. The 5' probe did not show hybridization in the heart and the lens RNAs. These data suggest that upstream initiations represent a minor population of transcripts and that higher size transcripts (about 500 bp larger) actually represent non-polyadenylated RNAs that may not contribute to the generation of the actual gene product.
Animals↗
DNA sequencing on minigels: an alternative technique for routine analyses.
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Cloning, Molecular↗
alpha A-crystallin is expressed in non-ocular tissues.
alpha-Crystallin, the predominant structural protein of the ocular lens, has been considered to be composed of two subunits, alpha A-crystallin and alpha B-crystallin. Of these two, alpha B-crystallin has been previously shown to be an extralenticular protein while alpha A-crystallin has been considered to be a lens-specific polypeptide. Using an antiserum directed against an N-terminal peptide of alpha-crystallin, we have detected a 20-kDa protein in various rat tissues including the brain, liver, lung, spleen, skin, and small intestine and in a number of established epithelial and fibroblast cell lines. PCR analysis of poly(A)-enriched RNA and Southern blot analysis indicated the presence of alpha A-crystallin mRNA sequences in different non-lenticular tissues. Among the non-ocular tissues examined, spleen showed the highest levels of alpha A-crystallin protein and mRNA. The identity of alpha A-crystallin sequences in the spleen was established by cloning and sequencing a polymerase chain reaction-amplified region of alpha A-crystallin mRNA. Sequences derived from spleen and eye revealed almost 100% identity at the nucleotide level. Interestingly, alpha A-crystallin and alpha B-crystallin seem to exist in an inverse quantitative relationship in the spleen and the heart, the two non-ocular tissues where they show highest concentrations, respectively. The known conserved evolution of alpha A-crystallin and the definitive demonstration of the non-ocular expression of this polypeptide suggest important non-crystallin functions for this protein.
Animals↗
Lens fiber cell differentiation and expression of crystallins in co-cultures of human fetal lens epithelial cells and fibroblasts.
Growth of the ocular lens is directed by the division and differentiation of a single layer of epithelial cells located at the equatorial region. It is conceivable that this region of the lens capsule presents a special microenvironment modulated by molecular cues emanating from the surrounding tissues. In an effort to investigate the source and nature of these molecular cues, we co-cultured human fetal lens epithelial cells and fibroblasts derived from the ciliary body. We observed morphological differentiation as evidenced by the appearance of differentiating lentoid structures associated with fibroblasts. Characterization of the expression of lens-specific proteins revealed that in addition to alpha B-crystallin, these lentoid structures contain the lens fiber cell-specific proteins, alpha A-crystallin, beta B2-crystallin and gamma S-crystallin. None of these crystallins could be found in the surrounding undifferentiated lens epithelial cells. Interestingly, alpha B-crystallin usually present in lens epithelial cells when cultured alone, was found to be markedly decreased, both in synthesis and content in the cells surrounding the differentiated structures, suggesting that the process of differentiation in vitro may concomitantly produce a factor(s) which modulates alpha B-crystallin expression in these cells.
Cell Differentiation↗
Alpha B-crystallin exists as an independent protein in the heart and in the lens.
Alpha B-crystallin, a polypeptide of molecular mass 22 kDa, is considered to be one of two subunits (alpha A and alpha B) of the multimeric lens-specific protein, alpha-crystallin. Recent demonstrations of the extra-lenticular presence of alpha B-crystallin have suggested that outside of the lens, this polypeptide may have functions independent of alpha A. Within the lens however, as part of the protein alpha-crystallin, its function is assumed to be structural. In an effort to investigate the functional status of alpha B-crystallin in the lens, we have characterized this polypeptide in the rat heart and the human lens. Unequivocal identity of alpha B-crystallin in the rat heart and the rat lens was established by the sequence analyses of the respective cDNA clones. Size exclusion chromatography (FPLC) and immunoblotting showed that in the rat heart, alpha B-crystallin exists as an aggregate of 300-400 kDa average molecular mass, similar to that of purified alpha B-crystallin isolated from bovine lens. Interestingly, analysis of the human lens proteins by immunoblotting showed that, with age, unlike alpha A-crystallin, the alpha B subunit remains detectable in the soluble fractions derived from normal lenses as old as 82 years. Importantly, the average molecular mass of the alpha B subunit in the soluble fractions prepared from 60-80-year-old human lens nuclei was also found to be 300-400 kDa. These data lead to the conclusion that alpha B-crystallin may exist as an independent protein not only in non-lens tissues (e.g. heart) but in the lens as well.
Aging↗
Detection of interphotoreceptor retinoid binding protein (IRBP) mRNA in human and cone-dominant squirrel retinas by in situ hybridization.
Interphotoreceptor retinoid binding protein (IRBP) is a soluble glycolipoprotein located between the neurosensory retina and pigment epithelium, which may serve to transport vitamin A derivatives between these tissues. The specific cell type responsible for IRBP synthesis has not been well established. To address this issue, we have examined the expression of IRBP mRNA in human and cone-dominant ground squirrel retinas by in situ hybridization. Optimal labeling and histological resolution were achieved with 35S- and 3H-labeled anti-sense riboprobes made from a human IRBP cDNA clone, and semi-thin wax-embedded retinal sections. In human retina, label was localized over the inner segments of both rod and cone photoreceptors. Quantitative analysis demonstrated a fourfold higher density of label over rod inner segments. In ground squirrel retina, labeling was found almost exclusively over the inner segments of cones. The results indicate that in human retina both rods and cones express IRBP mRNA, albeit at different levels. In cone-dominant species such as the ground squirrel, cones are the principal cell type responsible for IRBP mRNA synthesis.
Animals↗
Synthesis of nucleic acid probes on membrane supports: a procedure for the removal of unincorporated precursors.
We have used DNA bound to small pieces of nylon membrane for the synthesis of radioactive probes. The DNA to be used for generating the probe(s) is first bound to nylon membranes and then introduced into the reaction mix. The labeling reaction takes place on the membrane and therefore allows easy removal of unincorporated precursors by simple washing for 1-2 min. The clean labeled probe is eluted from the membrane in formamide or in water and is ready for use. This DNA-membrane can be stored for reuse. Synthesis of probes on a solid support such as nylon membrane thus circumvents problems associated with chromatographic manipulations needed for the separation of labeled DNA from unicorporated precursors. Probes synthesized in this manner are as efficient in detecting nucleic acid sequences as those synthesized in solution.
Blotting, Northern↗
Alpha B-crystallin is expressed in kidney epithelial cell lines and not in fibroblasts.
We have recently shown the presence of alpha B-crystallin in non-ocular tissues of diverse embryological origins such as the heart, brain, spinal cord, kidney, retina, etc. Using an alpha B-crystallin-specific antiserum and immunofluorescence, immunoblotting, immunoprecipitation and peptide mapping with Staphylococcus aureus protease, we demonstrate differential expression of alpha B-crystallin in epithelial and fibroblast cell lines. alpha B-Crystallin was detectable only in epithelial cell lines such as MDBK, MDCK, LLCPK1 and JTC-12, and was not observed in two kidney fibroblast cell lines, one skin fibroblast cell line, and one corneal fibroblast cell line. Differential expression of the alpha B-crystallin gene was also confirmed by Northern blot analysis of the RNAs isolated from these cell lines. These data suggest a cell-type-specific role for alpha B.
Animals↗
alpha B subunit of lens-specific protein alpha-crystallin is present in other ocular and non-ocular tissues.
alpha-Crystallin, a tissue specific structural protein of the ocular lens, is known to be composed of two subunits, alpha A and alpha B. By using a specific antibody in an immunoblotting procedure we have found that one of the subunits, alpha B is present in a number of non-lenticular tissues including the retina, heart, skeletal muscle, skin, brain, spinal cord and lungs. Interestingly, in the rat, this protein is present in significantly higher concentrations in adult than in fetal tissues and, with the exception of the lens, fetal and adult heart has the highest concentration among the tissues examined. That the protein in question is, in fact, alpha B, was confirmed a) by the remarkable similarity of Staphylococcus aureus protease peptide maps of the protein in the heart and purified alpha-crystallin and b) by the sequence analysis of a rat heart cDNA clone identified by the alpha B antibody. Based on these observations we conclude that while alpha A has a tissue-specific role, alpha B is a polypeptide of independent function not restricted to the ocular lens.
Animals↗