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The aim of our study was to obtain comprehensive insight into the bacteriological and clinical profile of community-acquired pneumonia requiring hospitalization. The patient population consisted of 100 patients admitted with the diagnosis of community-acquired pneumonia (CAP), as defined by British Thoracic society, from December 1998 to Dec 2000, at the Sher- i-Kashmir institute of Medical Sciences Soura, Srinagar, India. Gram negative organisms were the commonest cause (19/29), followed by gram positive (10/29). In 71 cases no etiological cause was obtained. Pseudomonas aeruginosa was the commonest pathogen (10/29), followed by Staphylococcus aureus (7/29), Escherichia coli (6/29), Klebsiella spp. (3/29), Streptococcus pyogenes (1/29), Streptococcus pneumoniae (1/29) and Acinetobacter spp. (1/29). Sputum was the most common etiological source of organism isolation (26) followed by blood (6), pleural fluid (3), and pus culture (1). Maximum number of patients presented with cough (99%), fever (95%), tachycardia (92%), pleuritic chest pain (75%), sputum production (65%) and leucocytosis (43%). The commonest predisposing factors were smoking (65%), COPD (57%), structural lung disease (21%), diabetes mellitus (13%), and decreased level of consciousness following seizure (eight per cent) and chronic alcoholism (one per cent). Fourteen patients, of whom, nine were males and five females, died. Staphylococcus aureus was the causative organism in four, Pseudomonas in two, Klebsiella in one, and no organism was isolated in seven cases. The factors predicting mortality at admission were - age over 62 years, history of COPD or smoking, hypotension, altered sensorium, respiratory failure, leucocytosis, and s0 taphylococcus pneumonia and undetermined etiology. The overall rate of identification of microbial etiology of community-acquired pneumonia was 29%, which is very low, and if serological tests for legionella, mycoplasma and viruses are performed the diagnostic yield would definitely be better. This emphasizes the need for further studies (including the serological tests for Legionella, mycoplasma and viruses) to identify the microbial etiology of CAP.

Submerged floating tunnels (SFTs) are innovative structural solutions to waterway crossings, such as sea-straits, fjords and lakes. As the width and depth of straits increase, the conventional structures such as cable-supported bridges, underground tunnels or immersed tunnels become uneconomical alternatives. For the realization of SFT, the structural response under extreme environmental conditions needs to be evaluated properly. This study evaluates the displacements and internal forces of SFT under hydrodynamic and three-dimensional seismic excitations to check the global performance of an SFT in order to conclude on the optimum design. The formulations incorporate modeling of ocean waves, currents and mooring cables. The SFT responses were evaluated using three different mooring cable arrangements to determine the stability of the mooring configuration, and the most promising configuration was then used for further investigations. A comparison of static, hydrodynamic and seismic response envelope curves of the SFT is provided to determine the dominant structural response. The study produces useful conclusions regarding the structural behavior of the SFT using a three-dimensional numerical model.

Background and Objective: Hepatitis virus infections are affecting millions of people worldwide, causing death, disability, and considerable expenditure. Chronic infection with hepatitis C virus (HCV) can cause severe public health problems because of their high prevalence and poor long-term clinical outcomes. Thus a fractional-order epidemic model of the hepatitis C virus involving partial immunity under the influence of memory effect to know the transmission patterns and prevalence of HCV infection is studied. Investigating the transmission dynamics of HCV makes the issue more interesting. The HCV epidemic model and worldwide dynamics are examined in this study. Calculate the basic reproduction number for the HCV model using the next-generation matrix technique. We determine the model's global dynamics using reproduction numbers, the Lyapunov functional approach, and the Routh-Hurwitz criterion. The model's reproduction number shows how the disease progresses., Methods: A fractional differential equation model of HCV infection has been created. Maximum relevant parameters, such as fractional power, HCV transmission rate, reproduction number, etc., influencing the dynamic process, have been incorporated. The model's numerical solutions are obtained using the fractional Adams method. Finally, numerical simulations support the theoretical conclusions, showing the great agreement between the two., Results: In the fractional-order HCV infection model, the memory effect, which is not seen in the classical model, was shown on graphs so that disease dynamics and vector compartments could be seen. We found that the fractional-order HCV infection model has more stages of freedom than regular derivatives. Fractional-order derivations, which may be the best and most reliable, explained bodily approaches better than classical order., Conclusion: The current study modeled and analyzed a fractional-order HCV infection model. The current approach results in a much better understanding of HCV transmission in a population, which leads to important insights into its spread and control, such as better treatment dosage for different age groups, identifying the best control measure, improving health, prolonging life, reducing the risk of HCV transmission, and effectively increasing the quality of life of HCV patients. The creation of a fractional-order HCV infection model, which provides a better understanding of HCV transmission dynamics and leads to significant insights for better treatment dosages, identification of optimal control measures, and ultimately improvement of the quality of life for HCV patients, is the study's major outcome., Competing Interests: Declaration of competing interest No conflict of interest exists in the submission of this manuscript, and the manuscript is approved by all listed authors for publication., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Thrombosis, a key factor in most cardiovascular diseases, is a major contributor to human mortality. Existing antithrombotic agents carry a risk of bleeding. Consequently, there is a keen interest in discovering innovative antithrombotic agents that can prevent thrombosis without negatively impacting hemostasis. Platelets play crucial roles in both hemostasis and thrombosis. We have previously characterized calcium- and integrin-binding protein 1 (CIB1) as a key regulatory molecule that regulates platelet function. CIB1 interacts with several platelet proteins including integrin α IIb β 3 , the major glycoprotein receptor for fibrinogen on platelets. Given that CIB1 regulates platelet function through its interaction with α IIb β 3 , we developed a fluorescence polarization (FP) assay to screen for potential inhibitors. The assay was miniaturized to 1536-well and screened in quantitative high-throughput screening (qHTS) format against a diverse compound library of 14,782 compounds. After validation and selectivity testing using the FP assay, we identified 19 candidate inhibitors and validated them using an in-gel binding assay that monitors the interaction of CIB1 with α IIb cytoplasmic tail peptide, followed by testing of top hits by intrinsic tryptophan fluorescence (ITF) and microscale thermophoresis (MST) to ascertain their interaction with CIB1. Two of the validated hits shared similar chemical structures, suggesting a common mechanism of action. Docking studies further revealed promising interactions within the hydrophobic binding pocket of the target protein, particularly forming key hydrogen bonds with Ser180. The compounds exhibited a potent antiplatelet activity based on their inhibition of thrombin-induced human platelet aggregation, thus indicating that disruptors of the CIB1- α IIb β 3 interaction could carry a translational potential as antithrombotic agents., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Bi-directional signaling through platelet integrin α IIb β 3 is essential in hemostasis and thrombosis. In quiescent platelets α IIb β 3 is in a low-affinity ligand binding state. However, upon platelet activation by agonists through inside-out signaling, a rapid switch in the conformation of the integrin results in a high affinity ligand binding state capable of binding soluble fibrinogen. Ligand binding to the α IIb β 3 induces a signaling termed outside-in signaling that regulate platelet spreading and clot retraction. These events are often interchangeably used to represent outside-in signaling pathway. Using pharmacological inhibitors of known signaling molecules that have been implicated to regulate outside-in signaling, we assessed human platelet spreading and clot retraction. We found that inhibition of PI3K, PLC, PKC, and FAK strongly attenuated both platelet spreading and clot retraction suggesting that they are essential for both clot retraction and platelet spreading. Whereas inhibition of Rac1, ROCK, p38, and MEK did not affect platelet spreading but significantly delayed clot retraction suggesting that these signaling molecules do not participate in platelet spreading. Interestingly, Src family kinases (SFKs) are required for platelet spreading and FAK activation but suppresses clot retraction since their inhibition causes faster clot retraction. Thus, it becomes evident that platelet spreading, and clot retraction are differently regulated through α IIb β 3 outside-in signaling and should not be used interchangeably as readout for α IIb β 3 outside-in signaling assessment. Significance Statement Current anti-platelet drugs have increased risk of bleeding and low efficacy. There is an increased effort to identify novel anti-platelet agents that have improved efficacy with reduced risk of bleeding. It is increasingly felt that inhibition of α IIb β 3- induced outside-in signaling may inhibit thrombosis without compromising hemostasis. However, the signaling entities regulating outside-in signaling is poorly understood. Our work included in this manuscript delineates the distinct signaling pathways involved in outside-in signaling and identify potential novel targets for intervention of thrombosis., (Copyright © 2024 American Society for Pharmacology and Experimental Therapeutics.)

On the global level, uranium is considered the main nuclear energy source, and its removal from terrestrial ores is enough to last until the end of the current century. Therefore, a major focus is attracted toward the capture of uranium from a sustainable source (seawater). Uranium recovery from seawater has been reported over the last few decades, and recently many efforts have been devoted to the preparation of such adsorbents with higher selectivity and adsorption capacity. The purpose of this review is to report the advancement in adsorbent preparation and modification of porous materials. It also discusses challenges such as adsorbent selectivity, low uranium concentration in seawater, contact time, biofouling, and the solution to the problems necessary to ensure a better adsorption performance of the adsorbent., Competing Interests: The author declares no competing financial interest., (© 2024 The Author. Published by American Chemical Society.)

The increasing demand for gold because of its high market price and its wide use in the electronic industry has attracted interest in gold recovery from electronic waste (e-waste). Gold is being dumped as solid e-waste which contains gold concentrations ten times higher than gold ores. Adsorption is a widely used approach for extracting gold from e-waste due to its simplicity, low cost, high efficiency, and reusability of adsorbent material. Natural polysaccharides received increased attention due to their natural abundance, multi-functionality, biodegradability, and nontoxicity. In this review, a brief history, and advancements in this technology were evaluated with recent developments in the preparation and mechanism advancements of natural polysaccharides for efficient gold recovery. Moreover, we have discussed some bifunctional modified polysaccharides with detailed gold adsorption mechanisms. The modified adsorbent materials developed from polysaccharides coupled with inorganic/organic functional groups would demonstrate an efficient technology for the development of new bio-based materials for efficient gold recovery from e-waste. Also, future views are recommended for highlighting the direction to achieve fast and effective gold recovery from e-waste in a friendly and sustainable manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Two of the most prevalent neurodegenerative disorders (NDDs), Alzheimer's disease (AD) and Parkinson's disease (PD), present significant challenges to healthcare systems worldwide. While the etiologies of AD and PD differ, both diseases share commonalities in synaptic dysfunction, thereby focusing attention on the role of neurotransmitters. The possible functions that platelets may play in neurodegenerative illnesses including PD and AD are becoming more acknowledged. In AD, platelets have been investigated for their ability to generate amyloid-ß (Aß) peptides, contributing to the formation of neurotoxic plaques. Moreover, platelets are considered biomarkers for early AD diagnosis. In PD, platelets have been studied for their involvement in oxidative stress and mitochondrial dysfunction, which are key factors in the disease's pathogenesis. Emerging research shows that platelets, which release glutamate upon activation, also play a role in these disorders. Decreased glutamate uptake in platelets has been observed in Alzheimer's and Parkinson's patients, pointing to a systemic dysfunction in glutamate handling. This paper aims to elucidate the critical role that glutamate receptors play in the pathophysiology of both AD and PD. Utilizing data from clinical trials, animal models, and cellular studies, we reviewed how glutamate receptors dysfunction contributes to neurodegenerative (ND) processes such as excitotoxicity, synaptic loss, and cognitive impairment. The paper also reviews all current medications including glutamate receptor antagonists for AD and PD, highlighting their mode of action and limitations. A deeper understanding of glutamate receptor involvement including its systemic regulation by platelets could open new avenues for more effective treatments, potentially slowing disease progression and improving patient outcomes.

Tile industry wastewater is known to contain a high concentration of TSS and turbidity resulting from various raw materials. In the present study, the effectiveness of the coagulation process on turbidity and TSS removal from Kuwait ceramic tile industry wastewater was investigated using ferric chloride as a coagulant. The experiments were conducted using jar tests to determine the optimum operating conditions of coagulant dosages, pH, and settling time. It was found that the coagulant dosage and medium pH greatly affect the efficiency of the coagulation process. A gradual increase in coagulant dosage from 10 to 50 mg/L increased the efficiency of turbidity removal from 95.6% to 99.5%. The efficiency of the coagulation process was also found to be dependent on pH values, where higher pH improved the efficiency of turbidity removal. It was found that a medium pH of 10, 1 h settling time, and 50 mg/L of coagulant dosage are the optimum process conditions to achieve almost complete removal of turbidity (99.5%) and TSS (99.8%). This study concluded that coagulation might be useful as a primary wastewater treatment process for tile industry wastewater.

COVID-19 is an infectious disease caused by the SARS-CoV-2 virus that caused an outbreak of typical pneumonia first in Wuhan and then globally. Although researchers focus on the human-to-human transmission of this virus but not much research is done on the dynamics of the virus in the environment and the role humans play by releasing the virus into the environment. In this paper, a novel nonlinear mathematical model of the COVID-19 epidemic is proposed and analyzed under the effects of the environmental virus on the transmission patterns. The model consists of seven population compartments with the inclusion of contaminated environments means there is a chance to get infected by the virus in the environment. We also calculated the threshold quantity R 0 to know the disease status and provide conditions that guarantee the local and global asymptotic stability of the equilibria using Volterra-type Lyapunov functions, LaSalle's invariance principle, and the Routh-Hurwitz criterion. Furthermore, the sensitivity analysis is performed for the proposed model that determines the relative importance of the disease transmission parameters. Numerical experiments are performed to illustrate the effectiveness of the obtained theoretical results., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

G protein-coupled receptors (GPCRs) mediate the majority of platelet activation in response to agonists. However, questions remain regarding the mechanisms that provide negative feedback toward activated GPCRs to limit platelet activation and thrombus formation. Here we provide the first evidence that GPCR kinase 6 (GRK6) serves this role in platelets, using GRK6-/- mice generated by CRISPR-Cas9 genome editing to examine the consequences of GRK6 knockout on GPCR-dependent signaling. Hemostatic thrombi formed in GRK6-/- mice are larger than in wild-type (WT) controls during the early stages of thrombus formation, with a rapid increase in platelet accumulation at the site of injury. GRK6-/- platelets have increased platelet activation, but in an agonist-selective manner. Responses to PAR4 agonist or adenosine 5'-diphosphate stimulation in GRK6-/- platelets are increased compared with WT littermates, whereas the response to thromboxane A2 (TxA2) is normal. Underlying these changes in GRK6-/- platelets is an increase in Ca2+ mobilization, Akt activation, and granule secretion. Furthermore, deletion of GRK6 in human MEG-01 cells causes an increase in Ca2+ response and PAR1 surface expression in response to thrombin. Finally, we show that human platelet activation in response to thrombin causes an increase in binding of GRK6 to PAR1, as well as an increase in the phosphorylation of PAR1. Deletion of GRK6 in MEG-01 cells causes a decrease in PAR1 phosphorylation. Taken together, these data show that GRK6 regulates the hemostatic response to injury through PAR- and P2Y12-mediated effects, helping to limit the rate of platelet activation during thrombus growth and prevent inappropriate platelet activation., (© 2020 by The American Society of Hematology.)

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that regulates activation of the c-Jun N-terminal kinase (JNK)- and p38-stress response pathways leading to apoptosis in nucleated cells. We have previously shown that ASK1 is expressed in platelets and regulates agonist-induced platelet activation and thrombosis. However, the mechanism by which platelet agonists cause activation of ASK1 is unknown. Here, we show that in platelets agonist-induced activation of p38 is exclusively dependent on ASK1. Both thrombin and collagen were able to activate ASK1/p38. Activation of ASK1/p38 was strongly dependent on thromboxane A2 (TxA2) and ADP. Agonist-induced ASK1 activation is blocked by inhibition of phospholipase C (PLC) β/γ activity or by chelating intracellular Ca2+. Furthermore, treatment of platelets with thapsigargin or Ca2+ ionophore robustly induced ASK1/p38 activation. In addition, calcium and integrin-binding protein 1 (CIB1), a Ca2+-dependent negative regulator of ASK1, associates with ASK1 in resting platelets and is dissociated upon platelet activation by thrombin. Dissociation of CIB1 corresponds with ASK1 binding to tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) and the autophosphorylation of ASK1 Thr838 within the catalytic domain results in full activation of ASK1. Furthermore, genetic ablation of Cib1 in mice augments agonist-induced Ask1/p38 activation. Together our results suggest that in resting platelets ASK1 is bound to CIB1 at low Ca2+ concentrations. Agonist-induced platelet activation causes an increase in intracellular Ca2+ concentration that leads to the dissociation of CIB1 from ASK1, allowing for proper dimerization through ASK1 N-terminal coiled-coil (NCC) domains., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The study reported CO 2 storage properties of single-walled carbon nanotubes (CNTs) modified by introducing amino groups onto CNT surfaces via a chemical process. Two different approaches were used to produce amino-functionalized nanotubes by adding lithium amide and l-lysine amino acid. Lithium amide was introduced on CNT surfaces, and then, it was further modified by adding amino-moiety (l-lysine amino acid) to obtain multiamino sites on the CNT surface for CO 2 storage. The aminated CNT were followed by CO 2 adsorption experiments, and amino group interactions with CO 2 have helped CNT to achieve higher adsorption capacity. The successful modification of CNTs showed ameliorated CO 2 storage capacity as compared to pristine CNT. The modified CNT possessed free amine groups on the surface, which led to an enhanced CO 2 adsorption capacity. The modified CNT samples were analyzed by X-ray photoelectron spectroscopy, infrared spectroscopy, and X-ray diffraction techniques., Competing Interests: The authors declare no competing financial interest.

Background: It is believed that activation of c-Src bound to the integrin β3 subunit initiates outside-in signaling. The involvement of αIIb in outside-in signaling is poorly understood., Objectives: We have previously shown that CIB1 specifically interacts with the cytoplasmic domain of αIIb and is required for αIIbβ3 outside-in signaling. Here we evaluated the role of CIB1 in regulating outside-in signaling in the absence of inside-out signaling., Methods: We used αIIb cytoplasmic domain peptide and CIB1-function blocking antibody to inhibit interaction of CIB1 with αIIb subunit as well as Cib1-/- platelets to evaluate the consequence of CIB1 interaction with αIIb on outside-in signaling., Results: Fibrinogen binding to αIIbβ3 results in calcium-dependent interaction of CIB1 with αIIb, which is not required for filopodia formation. Dynamic rearrangement of cytoskeleton results in CIB1-dependent recruitment of FAK to the αIIb complex and its activation. Disruption of the association of CIB1 and αIIb by incorporation of αIIb peptide or anti-CIB1 inhibited both FAK association and activation. Furthermore, FAK recruitment to the integrin complex was required for c-Src activation. Inhibition of c-Src had no effect on CIB1 accumulation with the integrin at the filopodia, suggesting that c-Src activity is not required for the formation of CIB1-αIIb-FAK complex., Conclusion: Our results suggest that interaction of CIB1 with αIIb is one of the early events occurring during outside-in signaling. Furthermore, CIB1 recruits FAK to the αIIbβ3 complex at the filopodia where FAK is activated, which in turn activates c-Src, resulting in propagation of outside-in signaling leading to platelet spreading.

Mitogen-activated protein kinases (MAPKs) are expressed in platelets and are activated downstream of physiological agonists. Pharmacological and genetic evidence indicate that MAPKs play a significant role in hemostasis and thrombosis, but it is not well understood how MAPKs are activated upon platelet stimulation. Here, we show that apoptosis signal-regulating kinase 1 (ASK1), a member of the MAP3K family, is expressed in both human and murine platelets. ASK1 is rapidly and robustly activated upon platelet stimulation by physiological agonists. Disruption of Ask1 ( Ask1 -/- ) resulted in a marked functional defect in platelets. Ask1 -/- platelets showed an impaired agonist-induced integrin α IIb β 3 activation and platelet aggregation. Although there was no difference in Ca 2+ rise, platelet granule secretion and thromboxane A 2 (TxA 2 ) generation were significantly attenuated in Ask1 -/- platelets. The defective granule secretion observed in Ask1 -/- platelets was a consequence of impaired TxA 2 generation. Biochemical studies showed that platelet agonists failed to activate p38 MAPK in Ask1 -/- platelets. On the contrary, activation of c-Jun N -terminal kinases and extracellular signal-regulated kinase 1/2 MAPKs was augmented in Ask1 -/- platelets. The defect in p38 MAPK results in failed phosphorylation of cPLA 2 in Ask1 -/- platelets and impaired platelet aggregate formation under flow. The absence of Ask1 renders mice defective in hemostasis as assessed by prolonged tail-bleeding times. Deletion of Ask1 also reduces thrombosis as assessed by delayed vessel occlusion of carotid artery after FeCl 3 -induced injury and protects against collagen/epinephrine-induced pulmonary thromboembolism. These results suggest that the platelet Ask1 plays an important role in regulation of hemostasis and thrombosis., (© 2017 by The American Society of Hematology.)

Fibrinogen binding to activated integrin induces outside-in signaling that results in stable platelet aggregates and clot retraction. How integrin αIIbβ3 is discouraged from spontaneous activation is not known. We have recently shown that junctional adhesion molecule-A (JAM-A) renders protection from thrombosis by suppressing integrin outside-in signaling. In this study, we show that JAM-A associates with integrin αIIbβ3 in resting platelets and dissociates upon platelet activation by agonists. We also show that integrin-associated JAM-A is tyrosine phosphorylated and is rapidly dephosphorylated upon platelet activation. C-terminal Src kinase (Csk) binds to tyrosine phosphorylated JAM-A through its Src homology 2 domain. Thus, JAM-A recruits Csk to the integrin-c-Src complex in resting platelets. Csk, in turn, keeps integrin-associated c-Src in an inactive state by phosphorylating Y(529) in its regulatory domain. Absence of JAM-A results in impaired c-SrcY(529) phosphorylation and augmentation of outside-in signaling-dependent c-Src activation. Our results strongly suggest that tyrosine-phosphorylated JAM-A is a Csk-binding protein and functions as an endogenous inhibitor of integrin signaling. JAM-A recruits Csk to the integrin-c-Src complex, where Csk negatively regulates c-Src activation, thereby suppressing the initiation of outside-in signaling. Upon agonist stimulation, JAM-A is dephosphorylated on the tyrosine, allowing the dissociation of Csk from the integrin complex, and thus facilitating outside-in signaling.

Transmigration of neutrophils (PMNs) from the vasculature into inflamed tissues, mediated by interactions between PMNs and adhesion molecules on endothelial cells, is an essential aspect of inflammation. The crucial adhesion molecules include junctional adhesion molecule (JAM)-A. Investigation of the role of this molecule in models of inflammatory disease has been limited, however, and results in different disease models have varied. No previous study has addressed JAM-A in lung disease or effects on oxidant stress and proinflammatory cytokines. We use JAM-A knockout mice and blocking antibodies to investigate the role of JAM-A in a murine model of acute lung injury (ALI). With either experimental system, we find that absence of JAM-A activity significantly reduces migration of PMNs into the alveolar space, with a resulting decrease in oxidative stress. However, there is no reduction in whole lung activity of PMN-associated myeloperoxidase, presumably reflecting the histologically observed retention of PMNs in lung tissue. Activity of these retained PMNs may account for our failure to find significant change in markers of lung oxidative stress or cytokine and chemokine levels in plasma, lung, and bronchoalveolar lavage fluid. We likewise see no JAM-A-related changes in markers of capillary permeability or lung injury. A similar lack of congruence between effects on PMN migration and tissue injury has been reported in other disease models and for other adhesion molecules in models of ALI. Our results thus confirm the crucial role of JAM-A in PMN transmigration but demonstrate that transmigration is not essential for other aspects of inflammation or for lung injury in ALI.

Deletion of the highly conserved gene for the major Ca(2+) efflux pump, Plasma membrane calcium/calmodulin-dependent ATPase 4b (Pmca4b), in the mouse leads to loss of progressive and hyperactivated sperm motility and infertility. Here we first demonstrate that compared to wild-type (WT), Junctional adhesion molecule-A (Jam-A) null sperm, previously shown to have motility defects and an abnormal mitochondrial phenotype reminiscent of that seen in Pmca4b nulls, exhibit reduced (P < 0.001) ATP levels, significantly (P < 0.001) greater cytosolic Ca(2+) concentration ([Ca(2+) ](c)) and ∼10-fold higher mitochondrial sequestration, indicating Ca(2+) overload. Investigating the mechanism involved, we used co-immunoprecipitation studies to show that CASK (Ca(2+) /calmodulin-dependent serine kinase), identified for the first time on the sperm flagellum where it co-localizes with both PMCA4b and JAM-A on the proximal principal piece, acts as a common interacting partner of both. Importantly, CASK binds alternatively and non-synergistically with each of these molecules via its single PDZ (PDS-95/Dlg/ZO-1) domain to either inhibit or promote efflux. In the absence of CASK-JAM-A interaction in Jam-A null sperm, CASK-PMCA4b interaction is increased, resulting in inhibition of PMCA4b's enzymatic activity, consequent Ca(2+) accumulation, and a ∼6-fold over-expression of constitutively ATP-utilizing CASK, compared to WT. Thus, CASK negatively regulates PMCA4b by directly binding to it and JAM-A positively regulates it indirectly through CASK. The decreased motility is likely due to the collateral net deficit in ATP observed in nulls. Our data indicate that Ca(2+) homeostasis in sperm is maintained by the relative ratios of CASK-PMCA4b and CASK-JAM-A interactions., (Copyright © 2011 Wiley Periodicals, Inc.)

Mounting evidence suggests that agonist-initiated signaling in platelets is closely regulated to avoid excessive responses to injury. A variety of physiologic agonists induce a cascade of signaling events termed as inside-out signaling that culminate in exposure of high-affinity binding sites on integrin α(IIb)β(3). Once platelet activation has occurred, integrin α(IIb)β(3) stabilizes thrombus formation by providing agonist-independent "outside-in" signals mediated in part by contractile signaling. Junctional adhesion molecule A (JAM-A), a member of the cortical thymocyte marker of the Xenopus (CTX) family, was initially identified as a receptor for a platelet stimulatory mAb. Here we show that JAM-A in resting platelets functions as an endogenous inhibitor of platelet function. Genetic ablation of Jam-A in mice enhances thrombotic function of platelets in vivo. The absence of Jam-A results in increase in platelet aggregation ex vivo. This gain of function is not because of enhanced inside-out signaling because granular secretion, Thromboxane A2 (TxA2) generation, as well as fibrinogen receptor activation, are normal in the absence of Jam-A. Interestingly, integrin outside-in signaling such as platelet spreading and clot retraction is augmented in Jam-A-deficient platelets. We conclude that JAM-A normally limits platelet accumulation by inhibiting integrin outside-in signaling thus preventing premature platelet activation.

Megakaryocytes are large, polyploid cells that produce platelets. We have previously reported that calcium- and integrin-binding protein 1 (CIB1) regulates endomitosis in Dami cells. To further characterize the role of CIB1 in megakaryopoiesis, we used a Cib1(-/-) mouse model. Cib1(-/-) mice have more platelets and BM megakaryocytes than wild-type (WT) controls (P < .05). Furthermore, subsequent analysis of megakaryocyte-CFU production revealed an increase with Cib1 deletion compared with WT (P < .05). In addition, BM from Cib1(-/-) mice, cultured with thrombopoietin (TPO) for 24 hours, produced more highly polyploid megakaryocytes than WT BM (P < .05). Subsequent analysis of TPO signaling revealed enhanced Akt and ERK1/2 phosphorylation, whereas FAK(Y925) phosphorylation was reduced in Cib1(-/-) megakaryocytes treated with TPO. Conversely, platelet recovery in Cib1(-/-) mice after platelet depletion was attenuated compared with WT (P < .05). This could be the result of impaired adhesion and migration, as adhesion to fibrinogen and fibronectin and migration toward an SDF-1α gradient were reduced in Cib1(-/-) megakaryocytes compared with WT (P < .05). In addition, Cib1(-/-) megakaryocytes formed fewer proplatelets compared with WT (P < .05), when plated on fibrinogen. These data suggest that CIB1 plays a dual role in megakaryopoiesis, initially by negatively regulating TPO signaling and later by augmenting proplatelet production.

Calcium- and integrin-binding protein 1 (CIB1) has been shown to be involved in cell spreading and migration. The signaling events regulated by CIB1 during cell migration are poorly understood. Here we found that accumulation of CIB1 at the tip of the filopodia requires an intact cytoskeleton. Depletion of CIB1 using shRNA affects formation of FAK- and phosphotyrosine-rich focal adhesions without affecting stress fiber formation. Overexpression of CIB1 results in cell migration on fibronectin and Erk1/2 MAP kinase activation. CIB1-induced cell migration is dependent upon Erk1/2 activation, since it is inhibited by the MEK-specific inhibitor PD98059. Furthermore, CIB1-induced cell migration, as well as Erk1/2 activation, is dependent on PKC, Src family kinases as well as PI-3 kinase as it is inhibited by bisindolylmaleimide 1, PP2, and wortmannin, respectively, in a dose-dependent manner. Co-expression of dominant-negative Cdc42 completely abolished CIB1-induced cell migration. Additionally, co-expression of constitutively active, but not dominant negative PAK1, a CIB1 binding protein, inhibited CIB1-induced cell migration. These results suggest that CIB1 positively regulates cell migration and is necessary for the recruitment of FAK to the focal adhesions. Furthermore, CIB1-induced cell migration is dependent on MAP kinase signaling and its function is attenuated by PAK1., (Copyright © 2011 Wiley Periodicals, Inc.)

Members of the polo-like kinases (Plk1, Plk2, Plk3 and Plk4) are involved in the regulation of various stages of the cell cycle and have been implicated in cancer progression. Unlike its other family members, the expression of Plk3 remains steady during cell cycle progression, suggesting that its activity may be spatiotemporally regulated. However, the mechanism of regulation of Plk3 activity is not well understood. Here, we show that calcium- and integrin-binding protein 1 (CIB1), a Plk3 interacting protein, is widely expressed in various cancer cell lines. Expression of CIB1 mRNA as well as protein is increased in breast cancer tissue as compared to normal tissue. CIB1 constitutively interacts with Plk3 as determined by both in vitro and in vivo assays. This interaction of CIB1 with Plk3 is independent of intracellular Ca(2+). Furthermore, binding of CIB1 results in inhibition of Plk3 kinase activity both in vitro and in vivo. Interestingly, this inhibition of the Plk3 activity by CIB1 is Ca(2+)-dependent. Taken together, our results suggest that CIB1 is a regulatory subunit of Plk3 and it regulates Plk3 activity in a Ca(2+)-dependent manner. Furthermore, upregulation of CIB1 in cancer cells could thus inhibit Plk3 activity leading to abnormal cell cycle regulation in breast cancer cells. Thus, in addition to Plk3, CIB1 may be a potential biomarker and target for therapeutic intervention of breast cancer.

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that are involved in the regulation of the various stages of the cell cycle. Plk2 and Plk3, two members of this family, are known to interact with calcium- and integrin-binding protein 1 (CIB1). Activity of both Plk2 and Plk3 is inhibited by CIB1 in a calcium-dependent manner. However, the physiological consequences of this inhibition are not known. Here, we show that overexpression of CIB1 inhibits T47D cell proliferation. Overexpression of CIB1 or knockdown of Plk3 using shRNA produced a multinucleated phenotype in T47D cells. This phenotype was not cancer cell specific, since it also occurred in normal cells. The cells overexpressing CIB1 appear to undergo proper nuclear division, but are unable to complete the process of cytokinesis, thus forming large multinucleated cells. Both CIB1 overexpression and Plk3 knockdown disrupted microtubule organization and centrosomal segregation, which may have led to incomplete cytokinesis. The observed effect of CIB1 overexpression is not due to the inhibition of Plk2 by CIB1. Plk3 and CIB1 both colocalize at the centrosomes, however, localization of CIB1 is dependent on the expression of Plk3. Furthermore, expression of Plk3 blocks the multinucleated phenotype induced by expression of CIB1 in these cells. These results suggest that CIB1 tightly regulates Plk3 activity during cell division and that either over- or underexpression results in a multinucleated phenotype., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Unlabelled: Agonist-induced inside-out signaling activates platelet integrin alpha(IIb)beta(3), rendering it to bind plasma fibrinogen (Fg). Fg binding induces outside-in signaling that culminates in platelet aggregation, leading to physiological hemostasis and pathological thrombosis. How outside-in signaling through alpha(IIb)beta(3) regulates hemostasis and thrombosis is not well understood. We have previously shown that CIB1 is involved in regulating alpha(IIb)beta(3) function., Objective: To determine the in vivo role of CIB1 in the process of hemostasis and thrombosis., Methods and Results: Genetic ablation of Cib1 significantly increased mouse tail bleeding time. Greater than 50% of the Cib1 null mice showed a rebleeding phenotype. Time taken for complete occlusion of carotid artery upon 10% FeCl(3)-induced injury was significantly delayed in the absence of Cib1. This was also associated with unstable thrombus formation. The inside-out signaling appears normal as ADP-, collagen- and PAR4 peptide-induced aggregation and fibrinogen binding was unaffected. The absence of Cib1 also affected the ability of platelets to spread on immobilized Fg, but not filopodia formation. Spreading could be restored in Cib1 null platelets by the addition of exogenous ADP. Outside-in signaling-dependent tyrosine phosphorylation of the integrin beta(3) subunit was significantly reduced in the absence of Cib1 as determined by Western blot analysis., Conclusion: Using gene knockout mice, we show for the first time that lack of Cib1 results in impaired thrombosis. CIB1 regulates these processes by affecting platelet spreading, but not platelet filopodia formation. These in vivo and in vitro results clearly show that CIB1 is a key regulator of thrombosis.

Junctional Adhesion Molecule A (JAM-A) is a member of the Ig superfamily of membrane proteins expressed in platelets, leukocytes, endothelial cells and epithelial cells. We have previously shown that in endothelial cells, JAM-A regulates basic fibroblast growth factor, (FGF-2)-induced angiogenesis via augmenting endothelial cell migration. Recently, we have revealed that in breast cancer cells, downregulation of JAM-A enhances cancer cell migration and invasion. Further, ectopic expression of JAM-A in highly metastatic MDA-MB-231 cells attenuates cell migration, and downregulation of JAM-A in low-metastatic T47D cells enhance migration. Interestingly, JAM-A expression is greatly diminished as breast cancer disease progresses. The molecular mechanism of this function of JAM-A is beyond its well-characterized barrier function at the tight junction. Our results point out that JAM-A differentially regulates migration of endothelial and cancer cells.

The metastatic potential of cancer cells is directly attributed to their ability to invade through the extracellular matrix. The mechanisms regulating this cellular invasiveness are poorly understood. Here, we show that junctional adhesion molecule A (JAM-A), a tight junction protein, is a key negative regulator of cell migration and invasion. JAM-A is robustly expressed in normal human mammary epithelium, and its expression is down-regulated in metastatic breast cancer tumors. In breast cancer cell lines, an inverse relationship between JAM-A expression and the ability of these cells to migrate on a collagen matrix was observed, which correlates with the known ability of these cells to metastasize. The T47D and MCF-7 cells, which migrate least, are found to express high levels of JAM-A, whereas the more migratory MDA-MB-468 cells have lower levels of JAM-A on the cell surface. MDA-MB-231 cells, which are highly migratory, express the least amount of JAM-A. Overexpression of JAM-A in MDA-MB-231 cells inhibited both migration and invasion through collagen gels. Furthermore, knockdown of JAM-A using short interfering RNAs enhanced the invasiveness of MDA-MB-231 cells as well as T47D cells. The ability of JAM-A to attenuate cell invasion correlated with the formation of increased numbers of focal adhesions and the formation of functional tight junctions. These results show for the first time that an immunoglobulin superfamily cell adhesion protein expressed at tight junctions could serve as a key negative regulator of breast cancer cell invasion and possibly metastasis. Furthermore, loss of JAM-A could be used as a biomarker for aggressive breast cancer.

The process of new blood vessel formation from pre-existing vessels is known as angiogenesis. This process is important, both during physiological processes such as development and during wound healing, as well as during pathological processes, such as cancer and arthritis. Understanding the molecular mechanism of the regulation of angiogenesis and the identification of the key players involved in this process may help identify new therapeutic targets to combat and control angiogenesis and hence arthritis and cancer. This review focuses on a recently identified novel cell adhesion molecule, Junctional Adhesion Molecule A, and its role in the process of regulating angiogenesis.

Objective: We have previously shown that JAM-A regulates fibroblast growth factor-2 (FGF-2)-induced endothelial cell morphology, proliferation, and migration. Whether JAM-A is involved in FGF-2-induced angiogenesis in vivo is not known. We used JAM-A null mice to conclusively determine the role of JAM-A in FGF-2-induced neovascularization., Methods and Results: We generated JAM-A null (JAM-A(-/-)) mice using gene trap technology. These mice, although viable and fertile, exhibited distorted Mendelian and sex ratios, suggesting partial embryonic lethality. Retinal fluorescein angiogram did not reveal any significant morphological differences in the vasculature of JAM-A(-/-) mice compared with wild-type (JAMA-A(+/+)) littermates. To evaluate the role of JAM-A in angiogenesis, we performed an aortic ring assay. FGF-2-induced microvessel growth was evident in aortic rings from JAM-A(+/+) mice, but FGF-2 failed to induce microvessel sproutings in aortic rings from JAM-A(-/-) mice. In a Matrigel plug assay, a known in vivo model for angiogenesis, we found that FGF-2 induced a robust vessel growth in JAM-A(+/+) mice, whereas FGF-2 failed to induce blood vessel formation in plugs from JAM-A(-/-) mice., Conclusions: Our results using JAM-A(-/-) mice presented here conclusively establish an essential role for JAM-A in FGF-2-induced angiogenesis.

Junctional adhesion molecule-A (JAM-A) is a member of the immunoglobulin superfamily, and is mainly expressed in the tight junctions of both epithelial and endothelial cells. We have recently shown that JAM-A is involved in basic fibroblast growth factor (bFGF)-induced angiogenesis. Here, we show that, when ectopically expressed in human umbilical vein endothelial cells (HUVECs), JAM-A induced enhanced cell migration on vitronectin, but had no effect on fibronectin. Use of antibodies that block integrin function indicated that the migration on vitronectin is specific to integrin alpha(v)beta(3) and not to integrin alpha(v)beta(5). JAM-A-induced migration was inhibited by anti-JAM-A antibody. Additionally, overexpression of a JAM-A cytoplasmic domain deletion mutant failed to induce HUVEC migration. Addition of phosphoinositide 3-kinase and protein kinase C inhibitors blocked JAM-A-induced migration, suggesting that these kinases act downstream of JAM-A. Immunoprecipitation analysis showed that JAM-A interacts with integrin alpha(v)beta(3), and this association was increased by engagement of the ligand-binding site of the integrin by Arg-Gly-Asp-Ser (RGDS) peptide. Furthermore, activation of both focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK) on vitronectin was enhanced by JAM-A overexpression but not by its cytoplasmic domain deletion mutant. Taken together, these results suggest that signaling through JAM-A is necessary for alpha(v)beta(3)-dependent HUVEC migration and implicate JAM-A in the regulation of vascular function.

Objective: Recently, we have shown that blocking of junctional adhesion molecule-1/A (JAM-1/A) inhibits basic fibroblast growth factor (bFGF)-induced angiogenesis. Because the process of endothelial cell proliferation is a key initial step of neovascularization, we studied the effect of functional knockdown of JAM-1 on human umbilical vein endothelial cell (HUVEC) adhesion and migration induced by bFGF., Methods and Results: We introduced small interfering RNAs specific to JAM-1 in HUVECs, stimulated them with bFGF, and studied the resultant adhesion and migration of these cells on vitronectin and fibronectin. We show that depletion of JAM-1 inhibits bFGF-induced HUVEC migration specifically on vitronectin. This inhibition is not attributable to the failure of junctional organization, because expression and distribution of other junctional proteins remained unaffected. This inhibition was in fact attributed to an inability of JAM-1-depleted HUVECs to adhere and spread on vitronectin. Furthermore, we find that JAM-1-depleted HUVECs failed to activate extracellular signal-related kinase (ERK) in response to bFGF treatment., Conclusions: Our results show that JAM-1 is required for the bFGF-induced ERK activation that leads to endothelial cell migration on vitronectin. These data thus implicate JAM-1 as an integral part of both bFGF and ERK signaling pathways in endothelial cells.

Platelet spreading on the subendothelium in response to vascular injury is fundamental to the regulation of physiologic hemostasis. Previously, we have shown that, when bound to glycoprotein IIb (GPIIb), calcium- and integrin-binding protein (CIB) regulates platelet spreading on immobilized fibrinogen (Fg). In this study, we investigated the signaling events that occur downstream of CIB in the absence of signaling that occurs as a result of granular secretion. Using Chinese hamster ovary (CHO) cells as a model, we demonstrate that CIB induces cell migration. Immunofluorescence analysis of CIB localization indicates that endogenous CIB accumulates in areas of focal adhesions, and its overexpression up-regulates the formation of focal adhesion complexes compared with control cells. Immunoprecipitation analysis indicates that CIB associates with focal adhesion kinase (FAK), a key regulator in focal complex formation, and up-regulates its activity. Overexpression of dominant-negative FAK, FRNK, along with CIB in CHO cells completely inhibits CIB-induced cell migration. Further, confirmation of these data in the platelet system indicates that CIB and FAK associate throughout all stages of platelet spreading but only on Fg binding to GPIIb/IIIa. Taken together, our results suggest that CIB regulates platelet spreading through the regulation of FAK activation.

Growth factor-induced neovascularization has received a great deal of attention because it is fundamental to the growth and metastasis of solid tumors. This multistep process requires extensive signaling through growth factor receptors and integrins. Among the integrins involved in this process, integrin alphavbeta3 is specific to basic fibroblast growth factor (bFGF)-induced angiogenesis. Here we show that junctional adhesion molecule 1/A (JAM-1/A) and alphavbeta3 form a complex in the absence of bFGF. JAM-1, which is normally localized at the cell-cell junctions of quiescent endothelial cells, redistributes to the cell surface on bFGF treatment. Blockage of the extracellular domain of JAM-1 inhibits bFGF-induced endothelial cell morphology, proliferation, and angiogenesis. Additionally, mutation in the JAM-1 cytoplasmic domain blocks bFGF-induced mitogen-activated protein (MAP) kinase activation and ablates its ability to induce endothelial cell tube formation, suggesting that signaling through JAM-1 is key to bFGF-induced signaling. Immunoprecipitation analysis suggests that bFGF signaling dissociates the JAM-1/ alphavbeta3 complex, allowing for signaling through JAM-1 and alphavbeta3. In addition, blockage of either JAM-1 or alphavbeta3 inhibits bFGF-induced MAP kinase activation. Thus, our results suggest that signaling through JAM-1 and alphavbeta3 is necessary for bFGF-induced angiogenesis.

Platelet spreading on immobilized fibrinogen (Fg) involves progression through a number of morphologic stages that, although distinctive, are not well understood mechanistically. Here we demonstrate that an association between GPIIb/IIIa and calcium- and integrin-binding protein (CIB) is required for the process of platelet spreading. Upon platelet adhesion to immobilized Fg, CIB localizes to the transiently formed filopodia and then redistributes diffusely along the membrane periphery of spread platelets. Immunoprecipitation analyses indicate that CIB and glycoprotein IIb/IIIa (GPIIb/IIIa) interact with each other as platelets adhere to immobilized Fg, and together they associate with the platelet cytoskeleton. Introduction of anti-CIB antibody or GPIIb cytoplasmic peptide into platelets blocks lamellipodia but not filopodia formation. GPIIb peptide-induced inhibition of platelet spreading is recovered by the incorporation of recombinant CIB protein, suggesting that interaction between CIB and GPIIb/IIIa is required for progression from filopodial to spread morphologies. Further, anti-CIB- or GPIIb peptide-induced inhibition of platelet spreading can be overcome by the addition of exogenous adenosine diphosphate (ADP). These data suggest that formation of the CIB-GPIIb/IIIa complex may be necessary for initiation of downstream signaling events, such as ADP secretion, that lead to platelet spreading.

We have previously reported the purification and characterization of a 32 kDa platelet surface glycoprotein that is recognized by the stimulatory monoclonal antibody, F11. The cDNA has been cloned and found to encode the human homolog of the murine junctional adhesion molecule, JAM; we therefore named this human homolog JAM-1. Northern blot analysis indicated that JAM-1 mRNA is expressed as multiple species, the predominant transcript being approximately 4.0 kb in size. Genetic mapping analysis using fluorescence in situ hybridization (FISH) showed that it is localized to chromosome 1q21.1-21.3. Recombinant JAM-1, when expressed in Chinese hamster ovary (CHO) cells, localized to the cell membrane with intense staining where two adjacent cells actually made contact with each other, suggesting that, similar to murine JAM, human JAM-1 may also localize at the cell-cell junction. In well-spread cells, JAM-1 co-localized with F-actin at the cell-cell contacts and at the membrane ruffles, but not at the stress fibers. Interestingly, JAM-1 localizes only to the cell-cell junctions formed by two transfected cells and not to the cell-cell junctions formed by a transfected cell with an untransfected cell, suggesting that JAM-1 may facilitate cell adhesion through homophilic binding. In addition, human platelets specifically bind to a monolayer of CHO cells expressing human JAM-1, further supporting homophilic interactions. The results presented here indicate that JAM-1, a receptor for a platelet-activating antibody, is the human homolog of the junctional adhesion molecule. JAM-1 is a single copy gene, which is constitutively expressed on various tissues and cells, and may be involved in cell to cell adhesion through homophilic interaction.

Protein kinase C (PKC) is a multigene family of at least ten isoforms, nine of which are expressed in brain (alpha, betaI, betaII, gamma, delta, straightepsilon, eta, zeta, iota/lambda). Our previous studies have shown that many of these PKCs participate in synaptic plasticity in the CA1 region of the hippocampus. Multiple isoforms are transiently activated in the induction phase of long-term potentiation (LTP). In contrast, a single species, zeta, is persistently activated during the maintenance phase of LTP through the formation of an independent, constitutively active catalytic domain, protein kinase Mzeta (PKMzeta). In this study, we used immunoblot and immunocytochemical techniques with isoform-specific antisera to examine the distribution of the complete family of PKC isozymes and PKMzeta in rat brain. Each form of PKC showed a widespread distribution in the brain with a distinct regional pattern of high and low levels of expression. PKMzeta, the predominant form of PKM in brain, had high levels in hippocampus, frontal and occipital cortex, striatum, and hypothalamus. In the hippocampus, each isoform was expressed in a characteristic pattern, with zeta prominent in the CA1 stratum radiatum. These results suggest that the compartmentalization of PKC isoforms in neurons may contribute to their function, with the location of PKMzeta prominent in areas notable for long-term synaptic plasticity., (Copyright 2000 Wiley-Liss, Inc.)

Protein kinase C (PKC) is a heterogeneous family of ten or more isoforms which plays an important role in neuronal signal transduction. Isoforms from all subclasses are prominently expressed in the rat hippocampus, as demonstrated by immunoblot with isozyme-specific antisera: Ca(2+)-dependent (alpha, beta I, beta II and gamma), Ca(2+)-independent (delta, epsilon and a newly characterized PKC related to eta) and atypical (zeta). In addition, the zeta isoform is also found as the free, constitutively active catalytic domain, protein kinase M zeta (PKM zeta). Two distinct patterns of expression of PKC isozymes in rat hippocampus are found during development from E18 to P28. PKC zeta, PKM zeta and PKC delta are present at birth and their expression does not increase postnatally. In contrast, the other isoforms are expressed only at low levels at birth and then increase in the first 4 weeks postnatally. These two patterns of expression suggest distinct functions for PKC isozymes during development.

Long-term potentiation in the CA1 region of the hippocampus, a model for memory formation in the brain, is divided into two phases. A transient process (induction) is initiated, which then generates a persistent mechanism (maintenance) for enhancing synaptic strength. Protein kinase C (PKC), a gene family of multiple isozymes, may play a role in both induction and maintenance. In region CA1 from rat hippocampal slices, most of the isozymes of PKC translocated to the particulate fraction 15 sec after a tetanus. The increase of PKC in the particulate fraction did not persist into the maintenance phase of long-term potentiation. In contrast, a constitutively active kinase, PKM, a form specific to a single isozyme (zeta), increased in the cytosol during the maintenance phase. The transition from translocation of PKC to formation of PKM may help to explain the molecular mechanisms of induction and maintenance of long-term potentiation.

We describe a new form of protein kinase C (PKC) with a molecular weight of 97 kDa, higher than the known forms of vertebrate PKC. This putative new high-molecular weight isoform, which we are calling PKC (HMW), is increased in the membrane fraction either upon application of phorbol esters or with afferent synaptic stimulation of Schaffer collaterals in hippocampal slices. The protein cross-reacts on immunoblot with affinity-purified polyclonal antiserum raised against a peptide derived from the carboxy-terminus of PKC eta; it does not cross-react, however, with antiserum against the amino-terminal region of PKC eta. In the tissues examined, PKC(HMW) is localized primarily in brain, in contrast to PKC eta, which is found predominantly in lung and skin.