M. Bagchi

Neovascularization following traumatic brain injury: possible evidence for both angiogenesis and vasculogenesis

Abstract Objective: Our goal was to characterize the angiogenic response following traumatic brain injury (TBI). Methods: Western analysis for vascular endothelial growth factor (VEGF) expression, double immunofluorescence labeling of endothelium and vascular endothelial growth factor receptor 2 (VEGFR2), bromodioxyuridine (BrdU) incorporation and measurement of capillary density, were all used to determine the temporal angiogenic response following TBI. Results: The angiogenic factors, VEGF and VEGFR2, increase following trauma. Capillary density increases and BrdU incorporation confirm the presence of newly formed vessels up to 48 hours post-injury. Discussion: Our results indicated that following TBI, there is a substantial increase in angiogenesis and based on morphologic characterization of BrdU-positive nuclei within the endothelium, we provide evidence for vasculogenesis following injury.

Heat shock proteins of adult and embryonic human ocular lenses

We investigated the presence and distribution of heat shock proteins, HSP‐70 [Horwitz, J. 1992. Proc Natl Acad Sci 89:10449–10453], HSP‐40, HSc‐70, HSP‐27, and αβ‐crystallin in different regions of adult and fetal human lenses and in aging human lens epithelial cells. This study was undertaken because heat shock proteins may play an important role in the maintenance of the supramolecular organization of the lens proteins. Human adult and fetal lenses were dissected to separate the epithelium, superficial cortex, intermediate cortex, and nucleus. The water soluble and insoluble protein fractions were separated by SDS–PAGE, and transferred to nitrocellulose paper. Specific antibodies were used to identify the presence of heat shock proteins in distinct regions of the lens. HSP‐70 [Horwitz, 1992 ], HSP‐40, and HSc‐70 immunoreactivity was mainly detected in the epithelium and superficial cortical fiber cells of the adult human lens. The small heat shock proteins, HSP‐27 and αβ‐crystallin were found in all regions of the lens. Fetal human lenses showed immunoreactivity to all heat shock proteins. An aging study revealed a decrease in heat shock protein levels, except for HSP‐27. The presence of HSP‐70 [Horwitz, 1992 ], HSP‐40, and HSc‐70 in the epithelium and superficial cortical fiber cells imply a regional cell specific function, whereas the decrease of heat shock protein with age could be responsible for the loss of optimal protein organization, and the eventual appearance of age‐related cataract. J. Cell. Biochem. 84: 278–284, 2002.

Induction of mitosis in the cultured rabbit lens initiated by the addition of insulin to medium KEI-4.

Abstract The epithelium of lenses cultured in KEI-4, a completely defined medium formulated with specific reference to the biochemistry and physiology of the rabbit lens, exhibits a pattern of cell division similar to that noted for the organ in situ. Initial fluctuations in mitotic activity occurred in the area of the germinative zone during the first 24 hr of culture. Mitosis decreased at 1 hr, was extremely low at 3 hr and returned to values comparable for lens in vivo by 22 hr. The precipitous drop in mitosis noted at 3 hr is in part attributable to the isolation of the lens from adjoining tissue. The addition of insulin to KEI-4 triggers a parasynchronous burst of DNA synthesis throughout the central lens epithelium. The activation requires the intact hormone; neither proinsulin nor the A and/or B chains of insulin, nor glucagon nor zine chloride can initiate mitosis. The gamma-globulin-rich fraction of rabbit serum can also stimulate mitosis. The addition of dibutyryl adenosine 3′:5′ cyclic monophosphate (DBcAMP) plus theophylline to KEI-4-insulin inhibits mitosis and prevents the cells from entering the synthetic phase of the cell cycle. Theophylline alone or DBcAMP alone brings about a 90% reduction in the insulin-induced mitotic responses. Lenses exposed to insulin show a marked increase in RNA synthesis and also exhibit an increased binding of tritiated actinomycin D at 1 and 3 hr of culture relative to KEI-4 controls. The hormone apparently activates the genome including those genes governing cell division. The system is amenable for long-term culture of the mammalian lens and since the constituents of the medium are known it should be possible to determine the factor(s) in the medium which, in conjunction with insulin, are needed for the induction of cell division.

Vimentin synthesis by ocular lens cells

A study of the synthesis of the intermediate filament protein, vimentin, is reported here. The following systems were examined: the epithelial cells of the organ-cultured rabbit lens, the epithelial and cortical fiber cells of the organ-cultured adult chicken lens and the epithelial cells of the rabbit lens grown in tissue culture. Vimentin is actively synthesized by all of the above mentioned cells.

Heat shock proteins of chicken lens

The presence of heat shock proteins HSP‐40, HSP‐70, and HSc‐70 in adult and embryonic chicken lenses were determined. The epithelium, cortex, and nucleus of adult chicken lens were separated and tested for the presence of heat shock proteins (hsps) by western blot, using specific antibodies for HSP‐40, HSP‐70, and HSc‐70. Water soluble (WSF) and water insoluble fractions (WIF) of embryonic chicken lenses were isolated and tested for the presence of HSP‐40, HSP‐70, and HSc‐70 by immunoblot. Embryonic chicken lens sections were also analyzed for the presence of heat shock proteins by immunoflorescence technique. Data obtained from these experiments revealed that HSP‐40, HSP‐70, and HSc‐70 are present in all areas of both adult and embryonic chicken lens. Presence of hsps protein in the deep cortex and nucleus is intriguing as no detectable metabolic activities are reported in this area. However it can be proposed that hsps HSP‐40, HSP‐70, and HSc‐70 can interact with protein of these areas and protect them from stress induced denaturation. J. Cell. Biochem. 82:409–414, 2001.

Calponin phosphorylation in cerebral cortex microvessels mediates sustained vasoconstriction after brain trauma

Abstract Objectives: The purpose of this study was to determine the molecular and biochemical changes in the contractile protein, calponin (Cp), which temporally coincide with a previously reported state of sustained contractility following traumatic brain injury (TBI). Methods: Double immunofluorescence, western analysis and two-dimensional non-equilibrium pH gradient gel electrophoresis (NEPHGE)/SDS-PAGE techniques were utilized to determine both the location and extent of Cp within smooth muscle cells (SM) and the phosphorylation state of Cp following TBI, as induced using a weight drop acceleration impact model. Results: Double immunofluorescence for Cp and SM indicate that following injury, Cp migrates from the cytosol to a location subjacent to the SM membrane. Western analysis revealed a significant increase in Cp protein expression following injury that was maintained up to 48 hours post-injury. Combined Western analysis and NEPHGE indicated that Cp is phosphorylated following TBI. Discussion: Cp migration and phosphorylation may underlie the mechanism for increased vasoreactivity leading to hypoperfusion following TBI.

ERM proteins of the lens.

Ezrin and radixin and protein 4.1 were detected in the lens of the eye. These proteins were mainly present in the young elongating cortical fiber cells and localized to the plasma membranes. Moesin was not detected. Ezrin, radixin, and protein 4.1 provide another means whereby actin is linked to the plasma membrane in addition to the known adherens junctions in the lens.

Associated proteins of lens adherens junction

Cytoplasmic proteins associated with adherens junctions were identified in the chicken ocular lens. The catenins, α, β, and γ, were present in epithelial and fiber cells, although their pattern of distribution changed with fiber cell differentiation. The sharp decline in α‐catenin with fiber cell formation and the increasing Triton‐insolubility of N‐cadherin suggests that another subtype of α‐catenin exists in the lens. J. Cell. Biochem. 86: 700–703, 2002.

Regulation of Sam68 activity by small heat shock protein 22

Sam68 associates with c‐Src kinase during mitosis. We previously demonstrated that Sam68 functionally replaces and/or synergizes with HIV‐1 Rev in rev response element (RRE)‐mediated gene expression and virus production. Furthermore, we reported that knockdown of Sam68 inhibited Rev‐mediated RNA export and it is absolutely required for HIV‐1 production. In the present study, we identified small heat shock protein, hsp22, as a novel interacting partner of Sam68. Hsp22 binds to Sam68 in vitro and in vivo. Overexpression of hsp22 significantly inhibits Sam68‐mediated RRE‐ as well as CTE (constitutive transport element)‐dependent reporter gene expression. Furthermore, exposing 293T cells to heat shock inhibits Sam68/RRE function by virtue of elevating hsp22. The critical domain of hsp22 that interacts with Sam68 resides between amino acids 62 and 133. Our studies provide evidence for the first time that hsp22 specifically binds to Sam68 and modulates its activity, thus playing a role in the post‐transcriptional regulation of gene expression. J. Cell. Biochem. 99: 1353–1362, 2006.

An intrinsic membrane glycoprotein of the lens

A major glycoprotein of relative molecular weight of 130,000 daltons of chick lens cell membranes was identified by Concanavalin-A staining and enriched for by Con A-Sepharose chromatography. Using both Con A binding and immunological analyses with a specific polyclonal antibody, the glycoprotein was detected in chick lens epithelium, annular pad, cortex and nucleus. Localization to the plasma membranes of lens cells was demonstrated by immunofluorescence. A similar protein, which cross-reacts with the antibody to the chick protein and also binds Con A was demonstrated in human and bovine lenses.