Balakrishna L. Lokeshwar

Bio: Balakrishna L. Lokeshwar is an academic researcher from Georgia Regents University. The author has contributed to research in topics: LNCaP & Metastasis. The author has an hindex of 23, co-authored 45 publications receiving 7410 citations. Previous affiliations of Balakrishna L. Lokeshwar include State University of New York System & University of Miami.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Abstract: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

Human prostate cells synthesize 1,25-dihydroxyvitamin D3 from 25- hydroxyvitamin D3

Abstract: Epidemiological and laboratory data support a role for vitamin D in the growth and differentiation of human prostatic cells. These findings prompted us to ask whether prostatic cells could convert 25-hydroxyvitamin D3 (25-OH-D3), the major circulating metabolite of vitamin D3, to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the hormonally active metabolite, in a manner similar to cultured human keratinocytes. Therefore, we investigated three well-characterized human prostate cancer cell lines, LNCaP, DU 145, and PC-3; two primary cultures of cells derived from noncancerous human prostates (one normal and one benign prostatic hyperplasia); and primary cultures of normal human keratinocytes for their ability to synthesize 1,25(OH)2D3. Assays were performed in the presence of 25-OH-D3 as the enzyme substrate and 1,2-dianilinoethane, an antioxidant and free radical scavenger, and in the presence and absence of clotrimazole, a cytochrome P450 inhibitor. DU 145 and PC-3 cells produced 0.31 +/- 0.06 and 0.07 +/- 0.01 pmol of 1,25(OH)2D3/mg protein/h, respectively. No measurable 1,25(OH)2D3 was detected in LNCaP cells. The normal and benign prostatic hyperplasia primary cultures and keratinocyte cultures produced 3.08 +/- 1.56, 1.05 +/- 0.31, and 2.1 +/- 0.1 pmol of 1,25(OH)2D3/mg protein/h, respectively, using a calf thymus receptor binding assay to measure 1,25(OH)2D3 in the presence of 1,2-dianilinoethane. The identity of the analyte as 1,25(OH)2D3 was supported by high performance liquid chromatography using [3H]25-OH-D3 as the enzyme substrate and a solvent system that is specific for 1,25(OH)2D3. The production of 1,25(OH)2D3 in the prostate cancer cell lines and in the primary cultures was completely inhibited in the presence of clotrimazole. This report demonstrates that two of three human prostate cancer cell lines, as well as primary cultures of noncancerous prostatic cells, possess 1alpha-hydroxylase activity and can synthesize 1,25(OH)2D3 from 25-OH-D3. Together with recent data indicating that 1,25(OH)2D3 inhibits the invasiveness of human prostate cancer cells (G. G. Schwartz et al., Cancer Epidemiol. Biomark. Prev., 6: 727-732, 1997), these data suggest a potential role for 25-OH-D3 in the chemoprevention of invasive prostate cancer.

Tear fluid gelatinase B activity correlates with IL-1α concentration and fluorescein clearance in ocular rosacea

Abstract: PURPOSE. To correlate tear fluorescein clearance with interleukin-1α (IL-1α) concentration and gelatinase B (matrix metalloproteinase [MMP]-9) activity in the tear fluid of patients with ocular rosacea and normal control subjects. METHODS. Gelatinase activity was evaluated by gelatin zymography in tear fluid obtained from 13 patients with ocular rosacea (including 1 patient with recurrent epithelial erosion, 2 with recurrent peripheral corneal infiltrates and vascularization, and 2 patients with epithelial basement membrane dystrophy) and 13 normal subjects with normal aqueous tear production and no irritation symptoms. Tear fluorescein clearance was evaluated by measuring fluorescence in tear fluid collected from the inferior meniscus 15 minutes after instillation of 5 μl of 2% Na-fluorescein with a CytoFluor II fluorometer. Pro-MMP-9 and IL-1α concentrations in the tear fluid were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS. Compared with normal control subjects, patients with ocular rosacea had a greater delay of tear fluorescein clearance (P < 0.001), a higher tear IL-1α concentration (P < 0.001), and a greater pro- gelatinase B (92 kDa) activity (P < 0.001) in their tear fluid. The 84-kDa active form of gelatinase B was observed in 46% of the rosacea tear samples and none of the controls. The zymographic results were confirmed by ELISA that showed a significantly greater concentration of pro-MMP-9 (92 kDa) in the tear fluid of rosacea patients than controls. Delayed tear clearance was correlated with elevated tear IL-1 α concentration (ρ=0.67, P < 0.001) and increased tear gelatinase B activity (ρ=0.84, P < 0.001). Tear IL-1α concentration was correlated with tear gelatinase B activity (ρ=0.58, P < 0.002). CONCLUSIONS. Gelatinase B (MMP-9) activity is greater in patients with ocular rosacea than in normal eyes. The majority of this activity is due to 92-kDa proform of this enzyme. This activity is correlated with delayed tear clearance and tear fluid concentration of interleukin-1α, a proinflammatory cytokine that has been reported to stimulate gelatinase B production. Elevated gelatinase B activity in ocular rosacea may be involved in the pathogenesis of the irritation symptoms, recurrent epithelial erosions, vascularization, and epithelial basement membrane dystrophy that develops in the corneas of patients with this condition.

A mouse model of keratoconjunctivitis sicca.

Abstract: Purpose To evaluate the effects of pharmacologic inhibition of aqueous tear production and desiccating environmental stress on aqueous tear production, tear clearance, corneal epithelial permeability, and conjunctival epithelial morphology, proliferation, and conjunctival goblet cell differentiation. Methods Aqueous tear production was inhibited by applying transdermal scopolamine (scop) patches to the depilated midtail of female MC, CBA mice. Desiccating environmental stress was created by placing mice in a hood with a continuous airflow blower. Aqueous tear production and volume, tear clearance, and corneal barrier function were compared in four experimental groups: untreated control mice, mice placed in the blower hood, mice treated with a scop patch, and mice treated with a scop patch and blower hood (scop patch + blower). Cotton threads were used to evaluate aqueous tear production and volume. Tear clearance was assessed by fluorometric measurement of collected tear fluid 15 minutes after instillation of 1% sodium fluorescein. Corneal epithelial barrier function was assessed by fluorometric measurement of carboxyfluorescein uptake. Conjunctival morphology and goblet cell density were evaluated in stained histologic sections. Conjunctival epithelial proliferation was assessed by bromodeoxyuridine (BrdU) labeling. Results Significant decreases in cotton thread wetting and tear clearance were observed in mice treated with a scop patch (P Conclusions Pharmacologic inhibition of tear production in mice is accompanied by ocular surface epithelial changes resembling human keratoconjunctivitis sicca (KCS) that are exacerbated by desiccating environmental stress. Cholinergic stimulated tear production appears to be essential for maintaining a healthy ocular surface.

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Abstract: Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012

Abstract: In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including 'apoptosis', 'necrosis' and 'mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.

High prevalence of vitamin D inadequacy and implications for health.

Abstract: During the past decade, major advances have been made in vitamin D research that transcend the simple concept that vitamin D is Important for the prevention of rickets in children and has little physiologic relevance for adults. Inadequate vitamin D, in addition to causing rickets, prevents children from attaining their genetically programmed peak bone mass, contributes to and exacerbates osteoporosis in adults, and causes the often painful bone disease osteomalacia. Adequate vitamin D is also important for proper muscle functioning, and controversial evidence suggests it may help prevent type 1 diabetes mellitus, hypertension, and many common cancers. Vitamin D inadequacy has been reported in approximately 36% of otherwise healthy young adults and up to 57% of general medicine inpatients in the United States and in even higher percentages in Europe. Recent epidemiological data document the high prevalence of vitamin D inadequacy among elderly patients and especially among patients with osteoporosis. Factors such as low sunlight exposure, age-related decreases in cutaneous synthesis, and diets low in vitamin D contribute to the high prevalence of vitamin D inadequacy. Vitamin D production from cutaneous synthesis or intake from the few vitamin D-rich or enriched foods typically occurs only intermittently. Supplemental doses of vitamin D and sensible sun exposure could prevent deficiency in most of the general population. The purposes of this article are to examine the prevalence of vitamin D inadequacy and to review the potential implications for skeletal and extraskeletal health.