Cryopreservation of chick islets.

The avian endocrine pancreas shares some similarities with those of mammals. Previously we have reported a technique of isolation of B islets from chick pancreas and also demonstrated their possible use for hypoglycemic drug screening as efficiently as mammalian islets. Here we describe a novel cryopreservative medium for the cryopreservation of chick islets. Isolated chick islets were suspended in a cryo medium consisting of Dulbecco's modified Minimum Essential Medium: Ham's F12 (1:1), Fetal Bovine Serum (20%), Dimethylsulfoxide (10%) with different concentrations (50 microg/ml to 500 microg/ml) of riboflavin or nicotinamide. The viability of revived islets after three and half months was checked by trypan blue dye exclusion and functionality was assessed by insulin secretion in response to glucose challenge. The maximum recovery of viable islets and insulin secretion was obtained in response to glucose challenge at 250 microg/ml concentration of Riboflavin or Nicotinamide. This is a first report on cryopreservation of chick islets exhibiting cryoprotective role of water soluble vitamins without vitamin C.

Islet-derived stellate-like cells as a novel source for islet neogenesis in chicks.

The therapeutic potential of stem cells has led to renewed interest in regenerative biology. Pancreatic stellate cells have been reported in the mammalian pancreas; however, there are very few reports on stellate cells in the chicken pancreas. The intercalated duct epithelial cells observed in the A and B islets of the chicken pancreas have been claimed to be stellate cells from their morphological appearance. While isolating islets and acinar cells from the chick pancreas, we have found a population of stellate-like cells, which has been successfully propagated in a defined nutrient medium. These cells were immunopositive for vimentin, desmin, and fibronectin and also expressed alkaline phosphatase, indicating their undifferentiated state. On exposure to serum-free medium containing specific nutrients and differentiating agents, these stellate-like-cells gave rise to islet-like cell clusters. Islet-like clusters stained positive for the islet specific stain diphenyl thiocarbazone and were immunopositive for C-peptide indicating de novo insulin synthesis. These clusters secreted insulin in response to glucose challenge, thus suggesting their similarity to islets. Thus stellate cells found in chick pancreatic islets exhibit potential to differentiate into islet-like clusters. Taken together, our study documents for the first time the presence of a stellate-like cell population in chick pancreatic islets providing a source for islet neogenesis.

Isolated islets in diabetes research.

This review highlights some recent developments and diversified applications of islets in diabetes research as they are rapidly emerging as a model system in biomedical and biotechnological research. Isolated islets have formed an effective in vitro model in antidiabetic drug development programme, screening of potential hypoglycaemic agents and for investigating their mechanisms of action. Yet another application of isolated islets could be to understand the mechanisms of beta cell death in vitro and to identify the sites of intervention for possible cytoprotection. Advances in immunoisolation and immunomodulation protocols have made xeno-transplantation feasible without immunosuppression thus increasing the availability of islets. Research in the areas of pancreatic and non pancreatic stem cells has given new hope to diabetic subjects to renew their islet cell mass for the possible cure of diabetes. Investigations of the factors leading to differentiation of pancreatic stem/progenitor cells would be of interest as they are likely to induce pancreatic regeneration in diabetics. Similarly search for the beta cell protective agents has a great future in preservation of residual beta cell mass left after diabetogenic insults. We have detailed various applications of islets in diabetes research in context of their current status, progress and future challenges and long term prospects for a cure.

Chick pancreatic B islets as an alternative in vitro model for screening insulin secretagogues.

Previously, we reported a simple technique to isolate functional B islets from chick pancreata with retention of their insulin secretory ability in response to glucose challenge. To test the hypothesis that chick B islets are equally good candidates as mammalian islets for screening hypoglycemics and insulin secretagogues, we compared the structural and functional status of chick B islets with those of normal and diabetic mouse islets. Pancreata from chick, normal (nondiabetic) mice, and diabetic mice were collected, fixed, and processed for histological analysis using Gomori stain to distinguish A and B cells from islets. Similarly isolated islets from these animals were treated with different concentrations of tolbutamide, a known insulin secretagogue, and glucose to study insulin release. Histological analysis of pancreata from chicks and normal mice revealed intact B cells, whereas those from diabetic mice were destroyed. The insulin secretory response of chick B islets against the tolbutamide and glucose challenge was comparable to that of normal mouse islets. However, diabetic mouse islets did not respond to glucose challenge, indicating impaired functionality. We have identified a critical window that lies within 5 to 6 d posthatching for isolating chick B islets showing maximum glucose responsiveness and insulin secretion. The previous reports on chicken pancreatic islets involve the use of 4- to 6-wk-old chicks in which islets were found to be nonresponsive to glucose and, hence, could not be used for testing insulin secretory activity. However, our data on B islets from 5- to 6-d-old chick pancreata is highly promising, as islets are responsive to insulin secretagogues. The present data thus indicates that chick B islets can be used as an alternative in vitro model for screening insulin secretagogue and hypoglycemics.

Papillary carcinoma in a giant thyroglossal duct cyst.

Thyroglossal duct cysts (TGDCs) are common, however, a malignancy occurring in a TGDC is rare. The presence on an underlying malignancy is clinically occult but may be detected on preoperative imaging studies. We describe the CT findings of a papillary carcinoma occurring in a TGDC.

Drosophila Cdk4 is required for normal growth and is dispensable for cell cycle progression.

Complexes of D-type cyclins and cdk4 or 6 are thought to govern progression through the G(1) phase of the cell cycle. In Drosophila, single genes for Cyclin D and Cdk4 have been identified, simplifying genetic analysis. Here, we show that Drosophila Cdk4 interacts with Cyclin D and the Rb homolog RBF as expected, but is not absolutely essential. Flies homozygous for null mutations develop to the adult stage and are fertile, although only to a very limited degree. Overexpression of inactive mutant Cdk4, which is able to bind Cyclin D, does not enhance the Cdk4 mutant phenotype, confirming the absence of additional Cyclin D-dependent cdks. Our results indicate, therefore, that progression into and through the cell cycle can occur in the absence of Cdk4. However, the growth of cells and of the organism is reduced in Cdk4 mutants, indicating a role of D-type cyclin-dependent protein kinases in the modulation of growth rates.

The Drosophila cyclin D-Cdk4 complex promotes cellular growth.

Mammalian cyclin D-Cdk4 complexes have been characterized as growth factor-responsive cell cycle regulators. Their levels rise upon growth factor stimulation, and they can phosphorylate and thus neutralize Retinoblastoma (Rb) family proteins to promote an E2F-dependent transcriptional program and S-phase entry. Here we characterize the in vivo function of Drosophila Cyclin D (CycD). We find that Drosophila CycD-Cdk4 does not act as a direct G(1)/S-phase regulator, but instead promotes cellular growth (accumulation of mass). The cellular response to CycD-Cdk4-driven growth varied according to cell type. In undifferentiated proliferating wing imaginal cells, CycD-Cdk4 caused accelerated cell division (hyperplasia) without affecting cell cycle phasing or cell size. In endoreplicating salivary gland cells, CycD-Cdk4 caused excessive DNA replication and cell enlargement (hypertrophy). In differentiating eyes, CycD-Cdk4 caused cell enlargement (hypertrophy) in post-mitotic cells. Interaction tests with a Drosophila Rb homolog, RBF, indicate that CycD-Cdk4 can counteract the cell cycle suppressive effects of RBF, but that its growth promoting activity is mediated at least in part via other targets.

Actinobacillus actinomycetemcomitans immunosuppressive protein is a member of the family of cytolethal distending toxins capable of causing a G2 arrest in human T cells.

We have previously shown that Actinobacillus actinomycetecomitans produces an immunosuppressive factor (ISF) capable of impairing human lymphocyte function by perturbing cell cycle progression. We now report that ISF is the product of the cdtB gene, one of three genes encoding the family of cytolethal distending toxins (Cdt). The ISF polypeptide exhibits >/=95% identity with Hemophilus ducreyi CdtB protein and

Inhibition of poly(ADP-ribose) polymerase rescues human T lymphocytes from methylmercury-induced apoptosis.

The objective of this investigation was to determine the role of poly(ADP-ribose) polymerase (PARP) in methylmercuric chloride (MeHgCl)-induced T-cell apoptosis. Following exposure of human T-cells to 2.5 microM MeHgCl, we observed PARP activation within 45 min. Maximal activation was observed at 90 min after MeHgCl treatment; thereafter, PARP activity declined. The loss in enzyme activity was coincidental with the cleavage of 116-kDa intact PARP protein to an 85-kDa fragment. To address the relationship between PARP activation and induction of apoptosis, we first examined the redox status of T cells treated with MeHgCl. We found that exposure of T cells to low concentrations of this toxicant resulted in decreased levels of reduced pyridine nucleotides and an increase in the relative amounts of oxidized flavoproteins. Thus, the possibility exists that activation of PARP leads to NAD+ depletion and thereby alters mitochondrial redox status. To determine if PARP activation is indeed part of the proapoptotic (destructive) response or a component of the antiapoptotic (protective) response, we employed two inhibitors: 3-aminobenzamide and nicotinamide. Pretreatment of T cells with these inhibitors protected cells from MeHgCl-induced apoptosis; this was seen as a reduction in the uptake of Hoechst 33258 and DNA fragmentation. Moreover, these inhibitors blocked MeHgCl-induced oxidative stress as evidenced by a reduction in reactive oxygen species (ROS) generation. These agents, however, failed to block MeHgCl-dependent decline in mitochondrial transmembrane potential (delta psi m). We conclude that PARP activation leads to proapoptotic events that contribute to MeHgCl-induced cell death.

Modulation of chondrocyte proliferation by ascorbic acid and BMP-2.

Chondrocytes show an unusual ability to thrive under serum-free conditions as long as insulin, thyroxine, and cysteine are present. Studies with sternal chondrocytes from chick embryos indicate that thymidine incorporation in chondrocytes cultured under serum-free conditions is 30-50% of that seen with fetal bovine serum (FBS). In contrast, skin fibroblast proliferation in serum-free culture is <5% of that seen with serum. Addition of 30-50 microM ascorbic acid to serum-free medium stimulates chondrocyte proliferation 4-5x, resulting in levels of thymidine incorporation higher than that seen with 10% serum. Three to five hours of ascorbate exposure is sufficient to stimulate proliferation, with maximal stimulation seen after 12-15 h. Bromo-deoxyuridine (BrdU) labelling indicated that approximately 25% of chondrocytes transit S phase during a 4-h period (16-20 h after ascorbate). Once maximal stimulation is reached, the proliferation rate remains fairly constant over at least 40 h. Ascorbate therefore increases the steady-state level of chondrocytes in the cycle. Because the stimulation of chondrocyte proliferation was greater than the net increase in cell numbers, we examined the level of apoptosis. Nuclear morphology, terminal uridine nucleotide end-labelling (TUNEL) assay, and 7-AAD/Hoechst dye FACS analyses all indicated that approximately 15% of the ascorbate-treated chondrocytes were undergoing apoptosis, while only 5% of the control chondrocytes were apoptotic. When prehypertrophic chondrocytes from the cephalic region of embryonic sternae were stimulated to undergo hypertrophy with rhBMP-2 + ascorbate, levels of apoptosis were similar to that seen with ascorbate alone. In contrast, treatment of caudal chondrocytes with BMP plus ascorbate does not induce hypertrophy, and the proportion of apoptotic cells was less than that seen with ascorbate alone. These results imply that in chondrocytes the transition to hypertrophy is associated with a decreased number of proliferating cells and a relatively high level of apoptosis.

Induction of apoptosis in human T-cells by organomercuric compounds: a flow cytometric analysis.

Although several lines of investigation demonstrate that many heavy metals are cytotoxic to host defense cells, the mechanism of killing is poorly understood. The major focus of this investigation was to determine if organic mercuric compounds kill human lymphocytes by inducing the cells to undergo apoptosis and to evaluate possible flow cytometric systems for assessing cell death. T-cells were exposed to 0.6-5 microM MeHgCl, EtHgCl, or PhHgCl for up to 24 hr and then analyzed by flow cytometry. Mercury-treated cells exhibited increased Hoechst 33258 and 33342 fluorescence while maintaining their ability to exclude the vital stain 7-AAD. Furthermore, T-cells exposed to mercury exhibited changes in light scatter patterns that included decreased forward light scatter and increased side scatter. The light scatter and fluorescent changes were consistent with changes that cells display during apoptosis. To further evaluate cell death and to distinguish between apoptosis and necrosis, merocyanine 540 staining and annexin V binding to the plasma membrane as well as DNA fragmentation were assessed. Mercury-treated cells exhibited increased merocyanine 540 fluorescence and annexin V binding along with changes in nuclear morphology consistent with the notion of apoptosis. Conventional agarose gel electrophoresis failed to demonstrate low-molecular-weight DNA bands; however, when probed by flow cytometry using both nick translation and a modified TUNEL assay, patterns consistent with nuclear fragmentation were evident. We noted that the percentage of T-cells undergoing apoptosis was dependent upon the amount of serum present in the medium; as serum concentrations were increased from 0 to 10%, cell death declined. Apoptosis (33%) was observed within 1 hr of exposure to MeHgCl; maximum cell death (67%) occurred after 24 hr exposure. Induction of apoptosis was dependent on the mercury concentration and independent of the hydrophobicity of the mercury ligand. Finally, we assessed mercury-dependent apoptosis in activated T-cells. When treated with mitogen, mercury failed to induce apoptosis in these cells. Indeed, there was no evidence of either apoptosis nor necrosis in these populations. It was concluded that the activation process prevented development of a metabolic state that was required for induction of apoptogenic genes.

Mercuric compounds inhibit human monocyte function by inducing apoptosis: evidence for formation of reactive oxygen species, development of mitochondrial membrane permeability transition and loss of reductive reserve.

The focus of this investigation was to examine the effects of low concentrations of organic mercuric compounds on human monocyte function and to relate these effects to apoptosis. Following exposure of monocytes to 0-5 microM MeHgCl, phagocytic function and capacity to generate a respiratory burst, following PMA activation, were determined. We found that the mercury-treated cells exhibited reduced phagocytic activity. Exposure to the same mercury concentration range, also caused a marked increase in cell death. To ascertain if monocyte death was due to apoptosis, a number of flow cytometric studies were performed. Mercury-treated cells exhibited increased Hoechst 33258 fluorescence, while maintaining their ability to exclude the vital dye 7-aminoactinomycin D. Furthermore, monocytes exhibited changes in light scatter patterns that were consistent with apoptosis; these included decreased forward light scatter and increased side scatter. The percentage of cells undergoing apoptosis was dependent upon the mercury content of the medium, regardless of whether the metal was present as methyl, ethyl or phenyl mercury. Mercury-treated cells also exhibited changes in lipid organization within the plasma membrane as evidenced by increased uptake of the fluorescent probe, merocyanine 540, and by elevated annexin V binding to phosphatidylserine. Using the fluorescent probes DiOC6(3) and rhodamine 123 we noted that within 1 h of exposure to mercury, monocytes exhibited a decrease in mitochondrial transmembrane potential (psi m). Since a decreased psi m is associated with altered mitochondrial function, the hypothesis that mercury potentiated reactive oxygen species (ROS) generation and that these species promoted apoptosis was tested. We noted that treated cells generated ROS, as evidenced by oxidation of hydroethidine and the generation of the fluorescent product, ethidium. Finally, since ROS would also lower monocyte reductive reserve, we also measured GSH levels in mercury-treated cells. Chemical measurement of GSH indicated that there was thiol depletion. We suggest that the low thiol reserve predisposes cells to ROS damage and at the same time activates death-signaling pathways.

Zygotic degradation of two maternal Cdc25 mRNAs terminates Drosophila's early cell cycle program.

In Drosophila embryos the maternal/zygotic transition (MZT) in cell cycle control normally follows mitosis 13. Here we show that this transition requires degradation of two maternal mRNAs, string and twine, which encode Cdc25 phosphatases. Although twine is essential for meiosis and string is essential for most mitotic cycles, the two genes have mutually complementing, overlapping functions in the female germ line and the early embryo. Deletion of both gene products from the female germ line arrests germ-line development. Reducing the maternal dose of both products can lower the number of early embryonic mitoses to 12, whereas increasing maternal Cdc25(twine) can increase the number of early mitoses to 14. Blocking the activation of zygotic transcription stabilizes maternal string and twine mRNAs and also allows an extra maternal mitosis, which is Cdc25 dependent. We propose that Drosophila's MZT comprises a chain reaction in which (1) proliferating nuclei deplete factors (probably mitotic cyclins) required for cell cycle progression; (2) this depletion causes the elongation of interphases and allows zygotic transcription; (3) new gene products accumulate that promote degradation of maternal mRNAs, including string and twine; and (4) consequent loss of Cdc25 phosphatase activity allows inhibitory phosphorylation of Cdc2 by Dwee1 kinase, effecting G2 arrest. Unlike timing or counting mechanisms, this mechanism can compensate for losses or additions of nuclei by altering the timing and number of the maternal cycles and thus will always generate the correct cell density at the MZT.

Fusobacterium nucleatum inhibits human T-cell activation by arresting cells in the mid-G1 phase of the cell cycle.

Fusobacterium nucleatum has been implicated in the pathogenesis of several diseases, including urinary tract infections, bacteremia, pericarditis, otitis media, and disorders of the oral cavity such as pulpal infections, alveolar bone abscesses, and periodontal disease. We have previously demonstrated that sonic extracts of F. nucleatum FDC 364 were capable of inhibiting human T-cell responses to mitogens and antigens. In this study, we have further characterized this immunosuppressive protein (FIP) and initiated experiments to determine its mode of action. The purified FIP has an apparent molecular mass of 90 to 100 kDa; sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the FIP is actually composed of two subunits with molecular masses of 48 and 44 kDa. Purified FIP retained its biological activity and was capable of inhibiting mitogen-induced proliferation of human T cells. Inhibition was dose dependent, and the FIP exhibited a specific activity approximately 250-fold greater than that of the crude extract. Cell cycle analysis indicates that FIP-treated cells were prevented from exiting the G0/G1 phase of the cell cycle. However, FIP did not alter the expression of activation markers (CD69, CD25, and CD71) or interleukin-2 secretion. The latter observations suggest that the T cells did indeed become activated and had entered the G1 phase of the cell cycle. Analysis of the expression of cyclins indicates that the phase of the cell cycle that is FIP sensitive resides somewhere beyond the restriction point of cyclin D2 (early to mid-G1) but prior to that of cyclins D3 and E (mid- to late G1). Finally, analysis of the expression of the proliferating cell nuclear antigen indicates that this is the earliest detectable defect in T cells exposed to FIP. We propose that if a block in the G1 phase of the cell cycle occurs in vivo in lymphocytes, it may result in a state of local and/or systemic immunosuppression. These suppressive effects could alter the nature and consequences of host-parasite interactions, thereby enhancing the pathogenicity of F. nucleatum itself or that of some other opportunistic organisms.

Testing the "redirection hypothesis" of prostaglandin metabolism in the kidney.

Furosemide increases the synthesis of two major renal eicosanoids, prostacyclin (PGI2) and thromboxane A2 (TXA2), by stimulating the release of arachidonic acid which in turn is metabolized to PGG2/PGH2, then to PGI2 and TXA2. PGI2 may mediate, in part, the early increment in plasma renin activity (PRA) after furosemide. We hypothesized that thromboxane synthetase inhibition should direct prostaglandin endoperoxide metabolism toward PGI2, thereby enhancing the effects of furosemide on renin release. Furosemide (2.0 mg . kg-1 i.v.) was injected into Sprague-Dawley rats pretreated either with vehicle or with U-63,557A (a thromboxane synthetase inhibitor, 2 mg/kg-1 followed by 2 mg/kg-1 X hr-1). Urinary 6ketoPGF1 alpha and thromboxane B2 (TXB2), reflecting renal synthesis of PGI2 and TXA2, as well as PRA and serum TXB2, were measured. Serum TXB2 was reduced by 96% after U-63,557A. U-63,557A did not affect the basal PRA. Furosemide increased PRA in both vehicle and U63,557A treated rats. However, the PRA-increment at 10, 20 and 40 min following furosemide administration was greater in U-63,557A-treated rats than in vehicle-treated rats and urine 6ketoPGF1 alpha excretion rates were increased. These effects of thromboxane synthesis inhibition are consistent with a redirection of renal PG synthesis toward PGI2 and further suggest that such redirection can be physiologically relevant.

Effect of cyclooxygenase and thromboxane synthetase inhibition on furosemide-stimulated plasma renin activity.

We studied the effects of a specific thromboxane (TX) synthetase inhibitor (U-63,557A) and a cyclooxygenase inhibitor on furosemide-induced renin release. Furosemide (2.0 mg X kg-1) was injected into Sprague-Dawley rats pretreated with indomethacin (10 mg X kg-1, i.v.), U-63,557A (1.0-32.0 mg X kg-1, i.v.), or vehicle (Na2CO3 0.03 M). Plasma renin activity was measured in blood samples collected 0, 10, 20, and 40 min after the injection of furosemide. Blood was also collected after the administration of vehicle, indomethacin, or U-63,557A for serum TXB2, a measure of platelet TXA2 synthesis. The results demonstrated that plasma renin activity rose with time following furosemide in the various groups of rats; indomethacin suppressed the furosemide-induced increments in plasma renin activity, while U-63,557A at doses of 4-8 mg X kg-1 augmented it. At doses below 4 mg X kg-1 or above 8 mg X kg-1, U-63,557A did not augment renin secretion. Indomethacin and U-63,557A reduced serum thromboxane by 81 and 90%, respectively. Thus, these experiments suggest that thromboxane synthetase inhibition, within a narrow dosage range, potentiates furosemide-induced renin release while cyclooxygenase inhibition suppresses it.

Clonidine fails to reduce pressor responsiveness of conscious spontaneously hypertensive rats to vasopressin.

Pressor responses and heart rate responses to intravenous injections (3.5-50.0 pmol/kg) of arginine vasopressin (AVP) were recorded in saline- and clonidine-treated spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. Clonidine (20 micrograms/kg, i.v.) caused a marked fall of arterial pressure in SHR but not in WKY rats so that, 20 min after the injection of the alpha 2-adrenoceptor agonist, arterial pressure was similar in the two strains of rats. The curve expressing the relationship between the dose of AVP and the increase of arterial pressure for saline-treated SHR was positioned to the left of that for saline-treated WKY rats. This enhanced pressor responsiveness of SHR to AVP may have been related to impaired reflex activity since heart rate fell much less in SHR than in WKY rats for a given elevation in pressure. Pressure responses to AVP were augmented by clonidine in both SHR and WKY rats so that, similar to saline-treated rats, pressor responsiveness to the peptide was still greater in SHR. Heart rate responses to AVP were not altered significantly by clonidine. The results indicate that clonidine fails to enhance reflex activity and reduce pressor responsiveness of SHR to AVP. The increased pressor responsiveness of both SHR and WKY rats to AVP following clonidine was an unexpected finding and may be related to a peripheral interaction between alpha-adrenergic agonists and AVP.

Reflexes fail to reduce pressor activity of vasopressin in spontaneous hypertension.

Pressor responses and heart rate responses were recorded in spontaneously hypertensive rats (SHR) and in Wistar-Kyoto (WKY) rats during intravenous infusions of arginine vasopressin and phenylephrine under two experimental situations, first when cardiovascular reflexes were impaired by pretreatment with a ganglionic blocking agent (pentolinium) and second, when reflexes remained intact. In rats with ganglia blocked, pressor responses of SHR to vasopressin or to phenylephrine were similar to those of WKY rats. In rats with intact reflexes, pressor responses of SHR to phenylephrine were also similar to those of WKY rats but, in contrast, pressor responses of SHR to vasopressin were enhanced. Heart rate fell much more for any given elevation of blood pressure in WKY rats than in SHR during infusions of vasopressin and phenylephrine, and the bradycardia associated with these pressor agents was largely abolished by pentolinium. The results are consistent with the hypothesis that baroreflexes buffer the pressor activity of vasopressin in the normotensive WKY rat and that an impairment of baroreflex activity in SHR contributes to the enhanced pressor activity of vasopressin in these rats.