TIME Impact - a new user-friendly tuberculosis (TB) model to inform TB policy decisions.

Tuberculosis (TB) is the leading cause of death from infectious disease worldwide, predominantly affecting low- and middle-income countries (LMICs), where resources are limited. As such, countries need to be able to choose the most efficient interventions for their respective setting. Mathematical models can be valuable tools to inform rational policy decisions and improve resource allocation, but are often unavailable or inaccessible for LMICs, particularly in TB. We developed TIME Impact, a user-friendly TB model that enables local capacity building and strengthens country-specific policy discussions to inform support funding applications at the (sub-)national level (e.g. Ministry of Finance) or to international donors (e.g. the Global Fund to Fight AIDS, Tuberculosis and Malaria).TIME Impact is an epidemiological transmission model nested in TIME, a set of TB modelling tools available for free download within the widely-used Spectrum software. The TIME Impact model reflects key aspects of the natural history of TB, with additional structure for HIV/ART, drug resistance, treatment history and age. TIME Impact enables national TB programmes (NTPs) and other TB policymakers to better understand their own TB epidemic, plan their response, apply for funding and evaluate the implementation of the response.The explicit aim of TIME Impact's user-friendly interface is to enable training of local and international TB experts towards independent use. During application of TIME Impact, close involvement of the NTPs and other local partners also builds critical understanding of the modelling methods, assumptions and limitations inherent to modelling. This is essential to generate broad country-level ownership of the modelling data inputs and results. In turn, it stimulates discussions and a review of the current evidence and assumptions, strengthening the decision-making process in general.TIME Impact has been effectively applied in a variety of settings. In South Africa, it informed the first South African HIV and TB Investment Cases and successfully leveraged additional resources from the National Treasury at a time of austerity. In Ghana, a long-term TIME model-centred interaction with the NTP provided new insights into the local epidemiology and guided resource allocation decisions to improve impact.

Whole-genome sequencing suggests a chemokine gene cluster that modifies age at onset in familial Alzheimer's disease.

We have sequenced the complete genomes of 72 individuals affected with early-onset familial Alzheimer's disease caused by an autosomal dominant, highly penetrant mutation in the presenilin-1 (PSEN1) gene, and performed genome-wide association testing to identify variants that modify age at onset (AAO) of Alzheimer's disease. Our analysis identified a haplotype of single-nucleotide polymorphisms (SNPs) on chromosome 17 within a chemokine gene cluster associated with delayed onset of mild-cognitive impairment and dementia. Individuals carrying this haplotype had a mean AAO of mild-cognitive impairment at 51.0 ± 5.2 years compared with 41.1 ± 7.4 years for those without these SNPs. This haplotype thus appears to modify Alzheimer's AAO, conferring a large (~10 years) protective effect. The associated locus harbors several chemokines including eotaxin-1 encoded by CCL11, and the haplotype includes a missense polymorphism in this gene. Validating this association, we found plasma eotaxin-1 levels were correlated with disease AAO in an independent cohort from the University of California San Francisco Memory and Aging Center. In this second cohort, the associated haplotype disrupted the typical age-associated increase of eotaxin-1 levels, suggesting a complex regulatory role for this haplotype in the general population. Altogether, these results suggest eotaxin-1 as a novel modifier of Alzheimer's disease AAO and open potential avenues for therapy.

Sarcoplasmic reticulum Ca(2+) atpase (SERCA) 1a structurally substitutes for SERCA2a in the cardiac sarcoplasmic reticulum and increases cardiac Ca(2+) handling capacity.

Ectopic expression of the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) 1a pump in the mouse heart results in a 2.5-fold increase in total SERCA pump level. SERCA1a hearts show increased rates of contraction/relaxation and enhanced Ca(2+) transients; however, the cellular mechanisms underlying altered Ca(2+) handling in SERCA1a transgenic (TG) hearts are unknown. In this study, using confocal microscopy, we demonstrate that SERCA1a protein traffics to the cardiac SR and structurally substitutes for the endogenous SERCA2a isoform. SR Ca(2+) load measurements revealed that TG myocytes have significantly enhanced SR Ca(2+) load. Confocal line-scan images of field-stimulated SR Ca(2+) release showed an increased rate of Ca(2+) removal in TG myocytes. On the other hand, ryanodine receptor binding activity was decreased by approximately 30%. However, TG myocytes had a greater rate of spontaneous ryanodine receptor opening as measured by spark frequency. Whole-cell L-type Ca(2+) current density was reduced by approximately 50%, whereas the time course of inactivation was unchanged in TG myocytes. These studies provide important evidence that SERCA1a can substitute both structurally and functionally for SERCA2a in the heart and that SERCA1a overexpression can be used to enhance SR Ca(2+) transport and cardiac contractility.

Ultrastructural distribution of NADPase within the Golgi apparatus and lysosomes of mammalian cells.

Cytochemical studies with over 40 different mammalian cell types have indicated that NADPase activity is associated with the Golgi apparatus and/or lysosomes of all cells. In the majority of cases, NADPase is restricted to saccular elements comprising the medial region of the Golgi stack and an occasional lysosome. There is often weak NADPase activity in other Golgi compartments such as the trans Golgi saccules and/or elements of the trans Golgi network. In some cells, however, strong NADPase activity is found within these latter compartments, either exclusively in trans Golgi saccules or elements of the trans Golgi network, or in combination with medial Golgi saccules and each other including (1) medial Golgi saccules + trans Golgi saccules, (2) medial Golgi saccules + trans Golgi saccules + trans Golgi network, or (3) trans Golgi saccules + trans Golgi network. In some rare cases, no NADPase activity is detectable in either Golgi saccules or elements of the trans Golgi network, but it is observed in an occasional lysosome or throughout the lysosomal system of these cells. It is unclear at present if these variations in the distribution of NADPase across the Golgi apparatus, and between the Golgi apparatus and lysosomal system, are due to differences in targeting mechanisms or to the existence of "bottlenecks" in the natural flow of NADPase along the biosynthetic pathway toward lysosomes. While no clear pattern in the association of strong NADPase activity with lysosomes was apparent relative to the ultrastructural distribution of NADPase activity in Golgi saccules or elements of the trans Golgi network, the results of this investigation suggested that cells having NADPase localized predominantly toward the trans aspect of the Golgi apparatus (in trans Golgi saccules or elements of the trans Golgi network or both) have few NADPase-positive lysosomes. The only exception is hepatocytes which were classified as predominantly trans but had noticeable NADPase activity within medial Golgi saccules and elements of the trans Golgi network as well, and highly reactive lysosomes. Other cells showing highly reactive lysosomes including (1) Kupffer cells of liver and those forming the proximal convoluted tubules of the kidney, both of which also had strong NADPase activity within medial and trans Golgi saccules and elements of the trans Golgi network, (2) Leydig cells of the testis and interstitial cells of the ovary, which also showed strong NADPase activity within medial Golgi saccules, and (3) macrophages from lung, spleen and testis, and Sertoli cells from the testis all of which showed no Golgi associated NADPase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Platelet-neutrophil interaction and superoxide anion generation: involvement of purine nucleotides.

Stimulated platelets secrete a variety of physiologically active substances that affect many neutrophil functions. We have examined the capacity of platelets to modulate superoxide anion generation by neutrophils. The amounts of superoxide anion produced by neutrophils in the presence of platelets were markedly enhanced when platelet-neutrophil coincubations were stimulated with agents that simultaneously activate both cell types, as the calcium ionophore A23187 and sodium arachidonate. This effect was dependent upon the number of platelets added to the incubation media and was not affected by inhibitors of arachidonic acid pathway or by preincubation of platelets with an antibody anti-P-selectin. The hypothesis of an involvement of purine nucleotides released by platelets during aggregation on the observed effect of enhancement of superoxide anion generation by neutrophils was then tested. Experimental evidence indicates that platelets release, during A23187-induced aggregation, amounts of ATP that are of the same order (5-10 microM) of those demonstrated to enhance superoxide anion generation by neutrophils. In addition, platelet lysates mimicked the effect of intact platelets in enhancing superoxide anion generation by A23187 stimulated neutrophils. Interestingly, at variance with the results obtained with intact platelets and platelet lysates, supernatants of thrombin-stimulated platelets did not increase O2.- by neutrophils. The enhancing effect of these supernatants was, however, restored when platelets were preincubated with an antibody anti P-selectin. These data indicate that platelets, through the release of purine nucleotides, enhance superoxide generation by neutrophils, thus increasing the cytotoxic potential of these cells.

Increased thrombogenic potential of human monocyte-derived macrophages spontaneously transformed into foam cells.

This study investigated whether spontaneous lipid enrichment of human macrophages affects their thrombogenic potential as measured by increased production of tissue factor (TF) and plasminogen activation inhibitor types 1 and 2 (PAI-1 and PAI-2). Macrophages were obtained following a 7-day culture period of monocytes, isolated from the same donor, in autologous serum (HS) or in fetal bovine serum (FBS). Those cultured in HS underwent marked lipid accumulation relative to those cultured in FBS that was accompanied by increased production of TF and PAI-1, but not of PAI-2, and decreased production of interleukin-1beta. They also contained more arachidonic and linoleic acid and lower amounts of n-3 polyunsaturated fatty acids, particularly docosahexaenoic acid (22: 6). These data indicate that the transformation of macrophages into foam cells results in an increase in their thrombogenic and antifibrinolytic potential and provide a possible explanation of the thrombotic sequelae frequently consequent on plaque fissuring and disruption.

Binding and internalization in vivo of [125I]hCG in Leydig cells of the rat.

The present study was performed to demonstrate the binding, mode of uptake, pathway and fate of iodinated human chorionic gonadotropin ([125I]hCG) by Leydig cells in vivo using electron microscope radioautography. Following a single injection of [125I]hCG into the interstitial space of the testis, the animals were fixed by perfusion with glutaraldehyde at 20 minutes, 1, 3, 6 and 24 hours. The electron microscope radioautographs demonstrated a prominent and qualitatively similar binding of the labeled hCG on the microvillar processes of the Leydig cells at 20 minutes, 1, 3, and 6 hours. The specificity of the [125I]hCG binding was determined by injecting a 100-fold excess of unlabeled hormone concurrently with the labeled hormone. Under these conditions, the surface, including the microvillar processes of Leydig cells, was virtually unlabeled, indicating that the binding was specific and receptor-mediated. In animals injected with labeled hCG and sacrificed 20 minutes later, silver grains were also seen overlying the limiting membrane of large, uncoated surface invaginations and large subsurface vacuoles with an electron-lucent content referred to as endosomes. A radioautographic reaction was also seen within multivesicular bodies with a pale stained matrix. At 1 hour, silver grains appeared over dense multivesicular bodies and occasionally over secondary lysosomes, in addition to the structures mentioned above, while at 3 and 6 hours, an increasing number of secondary lysosomes became labeled. At 24 hours, binding of [125I]hCG to the microvillar processes of Leydig cells persisted but was diminished, although a few endosomes, multivesicular bodies and secondary lysosomes still showed a radioautographic reaction. No membranous tubules that were seen in close proximity to, or in continuity with, endosomes and multivesicular bodies were observed to be labeled at any time interval. Likewise, an attempt to correlate silver grains with small coated or uncoated pits, the stacks of saccules of the Golgi apparatus and other Golgi-related elements including GERL, proved unsuccessful, since these structures were mostly unlabeled. These in vivo experiments thus demonstrate the specific binding of [125I]hCG to the plasma membrane of Leydig cells predominantly on their microvillar processes, and the subsequent internalization of the labeled hCG to secondary lysosomes. In addition, binding and internalization of hCG persisted for long periods of time.

Intracellular pathways of endocytosed tracers in Leydig cells of the rat.

The endocytic activity of Leydig cells was examined by electron microscopy following the injection, into the interstitial space, of tracers used to examine fluid-phase endocytosis, ie, native ferritin and horseradish peroxidase-colloidal gold (horseradish peroxidase-gold), and adsorptive endocytosis, ie, cationic ferritin. At 5 minutes after injection, native ferritin or horseradish peroxidase-gold was present in the interstitial space and free in the lumen of large endocytic vesicles forming at the cell surface. At 15 minutes, these tracers appeared in the matrix of pale multivesicular bodies, while at 30 minutes and 1 hour, the matrix of dense multivesicular bodies became labeled. Beginning at 1 hour, dense membrane-delimited bodies identified cytochemically as lysosomes were labeled. In the case of cationic ferritin, two distinct pathways were taken. In one pathway, cationic ferritin was observed 5 minutes after injection bound to the plasma membrane of Leydig cells and to the membrane of small and large endocytic vesicles. At subsequent time intervals, cationic ferritin appeared consecutively in pale, dense multivesicular bodies and lysosomes. In a second pathway, cationic ferritin was observed at 5, 15, and 30 minutes bound to the membrane of vesicles of intermediate size seen near the cell surface. At 1, 1 1/2 and 2 hours, cationic ferritin-containing intermediate vesicles appeared in increasing number in the Golgi region. However, cationic ferritin was never observed in the Golgi saccules themselves. At later time intervals (3-6 hours), intermediate vesicles labeled with cationic ferritin progressively disappeared from the Golgi region and the cell. Thus in Leydig cells, while fluid-phase tracers reached lysosomes exclusively, cationic ferritin, a tracer of adsorptive endocytosis, not only reached the lysosomes, but was also carried by the intermediate vesicles to the Golgi region of the cell.

Ultrastructure of fresh and frozen-thawed spermatozoa of high and low fertility lines of chickens.

Level of damage caused by freezing and thawing to four spermatozoal organelles (individual mitochondrion, midpiece, nucleus, and perforatorium) and the relationships of the integrity of these organelles in fresh and frozen-thawed semen with fertility were examined. Semen sample from 10th generation males of a line of chickens selected for increased duration of fertility of frozen-thawed semen and the corresponding randombred control line were used. In both the selected and control lines, the freeze-thaw process caused significant (P less than .05) detrimental damage to the ultrastructure of the mitochondria, midpiece, and perforatorium but not to the nucleus. Types of damage were identical in both lines. Granulated nuclei were observed in both frozen-thawed and freshly ejaculated spermatozoa and were referred to as a nuclear defect. This nuclear defect was associated with reduced fertility, the effect being more severe with frozen-thawed semen. Where the incidence of the nuclear defect was greater than 2% in frozen-thawed semen, fertility was found to be very low or nil regardless of the degree of structural integrity of the mitochondria, midpiece and perforatorium. Highly significant (P less than .01) positive correlation coefficients were observed for percentage fertility 2 to 8 days postinsemination and duration of fertility in days with percentages of normal mitochondria (.80 and .92), midpiece (.79 and .87), nucleus (.86 and .94), and perforatorium (.84 and .97) for fresh semen. With frozen-thawed semen, the positive correlation coefficients were significant (P less than .05) for midpiece (.64 and .69) and nucleus (.63 and .71) and nonsignificant for mitochondria (.52 and .50) and perforatium (.20 and .30).(ABSTRACT TRUNCATED AT 250 WORDS)

Structural changes of the head components of the rat spermatid during late spermiogenesis.

The transformation of the nucleus, acrosomic system, and peri-nuclear theca (perforatorium and post-acrosomal dense lamina) was analyzed during the maturation phase, i.e., steps 14 to 19 of spermiogenesis. Following partial condensation of chromatin from steps 11-14, the nucleus continues to condense during the following steps until the end of spermiogenesis. The redundant nuclear envelope which forms along the apical and ventral aspects of the nucleus and around the implantation fossa regresses during steps 17-19. The acrosomic system splits into two portions early in step 15 to give rise to: a) the main portion with its crest-like acrosome running along the dorsal aspect of the nucleus and head cap extending over the lateral surfaces of the nucleus: and b) a smaller head-cap segment which is seen in steps 15 and 16 along one side of the nucleus at its apical extremity. This separated head-cap segment reaches the apical-ventral aspect of the head during step 17 and condenses in synchrony with the rest of the acrosomic system in step 19 of spermiogenesis. The large crescentic acrosome, which in step 15 forms a large fin at the caudal extremity of the acrosomic apparatus, moves anteriorly during steps 16 and 17, while the whole acrosomic system extends farther apically beyond the tip of the nucleus. The perforatorium and post-acrosomal dense lamina form a rigid capsule (perinuclear theca) that covers tightly the sickle-shaped nucleus and binds the inner acrosomal membrane and the post-acrosomal membranes. The post-acrosomal dense lamina, which includes the ventral spur, appears early in step 15 as a dense cytoplasmic layer applied to the nuclear envelope at the caudal extremity of the nucleus except over the perifossal zone. The perforatorium forms during step 19 of spermiogenesis as a result of the condensation of a wispy cytoplasmic material which has accumulated in the subacrosomal space during steps 14-18. Thus the spermatid's head is deeply modified during the maturation phase and takes its definitive shape only at the last step of spermiogenesis.

Ultrastructural localization of nicotinamide adenine dinucleotide phosphatase (NADPase), thiamine pyrophosphatase (TPPase), and cytidine monophosphatase (CMPase) in the Golgi apparatus of early spermatids of the rat.

At the early steps 3-7 of spermiogenesis the hemispherical Golgi apparatus elaborates and is closely associated to the acrosomic system which grows at the surface of the spermatid's nucleus. It shows two distinct zones, a cortex made up of flattened saccules and related membranous tubules, and a medulla containing various types of vesicular profiles. The various components of the cortex of the Golgi apparatus were tested for their reactivity to three phosphatases. Nicotinamide adenine dinucleotide phosphatase activity (NADPase, Smith, 1980) was observed in the middle two to six saccules in the stack with a midsaccule being more reactive than the saccules above and below. A weak and spotty reaction was also noted in the remaining saccules on the trans-face of the stack and in the thick elements making up the GERL on the trans aspect of the stacks of saccules. Thiamine pyrophosphatase activity (TPPase, Novikoff and Goldfisher, 1961) was found in one or two saccules on the trans-face of the stacks but was absent from the other Golgi components. Cytidine monophosphatase activity (CMPase, Novikoff, 1967) was observed in the GERL, in vesicles of the medulla and in the developing acrosomic system. In the intersaccular regions of the cortex the branching membranous tubules showed the same reactivity for the phosphatases to that of the saccules to which they are connected. ER cisternae associated with the Golgi apparatus, anastomotic membranous tubules seen in the peripheral Golgi region, small vesicles, as well as the first saccule on the cis-face of the stacks were all negative for the three enzymes studied. These data indicated that in the cortex of the Golgi apparatus there were several distinct compartments that could be distinguished on the basis of structural and cytochemical features.

Arrangement of connective tissue components in the walls of seminiferous tubules of man and monkey.

In primates the membrane separating the seminiferous epithelium from the interstitial space is composed of one to three (monkey) or two to six layers (man) of myoid cells associated with one to two layers of fibrocyte-like adventitial cells. All these cells are separated from each other by irregular spaces filled with various connective tissue intercellular components. Subjacent to the elements of the seminiferous epithelium is a continuous, often redundant, basement membrane. A similar basement membrane-like material forms a layer next to and over small areas of the plasma membrane of myoid cells. Collagen fibrils grouped in bundles of various sizes are seen in all connective tissue layers but are particularly abundant in the space between the seminiferous epithelium and the innermost layer of myoid cells. Elastic fibrils demonstrated by the Verhoeff iron hematoxylin technique are also present. Composed of a homogeneous material, the elastic fibrils are short, irregular, branching entities with a diameter comparable to or smaller than that of collagen fibrils. In addition, an abundance of microfibrils with a diameter of 12-15 nm is present in the various connective tissue layers. These microfibrils have a densely stained cortex and a lightly stained core. When seen close to the myoid cells, bundles of micro fibrils appear to insert on well defined areas next to the plasma membrane. These areas commonly face the patches of electron-dense material observed on the inner aspect of the plasma membrane of the myoid cells and in which the actin filaments are inserted. Bundles of microfibrils often span the gap between myoid cells of the same layer as well as those of adjacent layers. Microfibrils are also closely related to the surface of elastic fibrils and are seen intertwining with collagen fibrils. Thus microfibrils appear to bridge and bind together adjacent myoid cells and anchor the surface of these cells to the bundles of elastic and collagen fibrils present in the intercellular spaces of the limiting membrane.

Evolution of the endoplasmic reticulum during spermiogenesis of the rooster: an electron microscopic study.

The endoplasmic reticulum (ER) of rooster's spermatids was analyzed during spermiogenesis, which was subdivided into eight distinct steps on the basis of changes observed with the electron microscope in the nucleus, acrosome-perforatorium system, manchette, and flagellum. In steps 1 and 2, spermatids' ER cisternae presented the following specializations: A loose network of tubular cisternae was distributed throughout the cytoplasm. Six to eight tight networks of anastomosed tubular cisternae parallel to each other were closely stacked to form a discoid body (1.5-2.5 microns in diameter and 0.5-0.8-micron thick) in which spheroidal vesicles (0.4 micron in diameter) were inserted. Close to and connected with this body, called the alveolar body, there was a stack of annulate lamellae. Large, flattened ER cisternae were seen singly or in piles of two or three running parallel to the nuclear surface. A collection of tubular ER cisternae faced plaques of thickened plasma membranes. These elements of the ER system appear continuous with each other. During steps 3-5 of spermiogenesis, no modification of the alveolar body-annulate lamellae complex was noted; the large flattened ER cisternae disappeared, however, and the broad network of tubular cisternae developed markedly. During steps 6 and 7, the latter network of tubular cisternae fragmented into vesicles that swelled to give a vacuolated appearance to the cytoplasm. The alveolar body-annulate lamellae complex remained visible until late step 7, when it disintegrated just before spermiation. Thus the system of ER cisternae underwent marked structural modifications during spermiogenesis.

Vanadate oxidation activates contraction in skinned smooth muscle without myosin light chain phosphorylation.

Phosphorylation of the myosin regulatory light chain (LC20-P1) is the major route of smooth muscle activation. However, after prior exposure to vanadate, permeabilized guinea pig taenia coli smooth muscle contracts in the absence of LC20-P1. We characterized the vanadate-induced contraction and investigated the mechanism of this novel activation pathway. Addition of vanadate to a control contracture (6.6 microM Ca2+) inhibits force (effective dose for 50% response was approximately 100 microM). In contrast, preincubation with high concentrations of vanadate (threshold at 1-2 mM) elicited a contraction on subsequent transfer of the fiber to a vanadate-free, Ca(2+)-free solution. Maximum isometric force of approximately 60% of control was obtained in fibers preincubated in 4 mM vanadate for 10 min. Addition of Ca2+ to a vanadate-induced contracture increased force, but the total force never exceeded the initial control. After maximal thiophosphorylation of LC20 with adenosine 5'-O-(3-thiotriphosphate), treatment with vanadate did not increase force. Unloaded shortening velocity (Vmax) was similar in Ca2+ and vanadate contractures and was additive. After thiophosphorylation, preincubation in vanadate had no effect on Vmax, suggesting that vanadate affected the number of activated bridges and not cycle rate. Vanadate mechanisms likely involve oxidation, since preincubation with 4 mM vanadate and 25 mM dithiothreitol (DTT) did not produce force. DTT could reverse a vanadate-induced contracture in 30-60 min. Subsequently, fibers demonstrated control contraction/relaxation cycles. Thus vanadate treatment did not cause irreversible damage, such as the extraction of proteins. Potential oxidation sites are proteins at 17 kDa and between 30 and 40 kDa, which were not alkylated by N-ethylmaleimide if they were treated in the presence of vanadate or in the rigor state. Vanadate-induced contractures are likely mediated by a reversible oxidation that activates cross bridges similarly to that of LC20-Pi and may play an important role in oxidant injury.

A cytochemical study of the Golgi apparatus of the spermatid during spermiogenesis in the rat.

The reactivity of the various components of the Golgi apparatus of rat spermatids for three phosphatase activities (nicotinamide adenine dinucleotide phosphatase, NADPase; thiamine pyrophosphatase, TPPase; cytidine monophosphatase, CMPase) and the incorporation of 3H-fucose by the spermatids was analyzed at the 19 steps of spermiogenesis, i.e., during and after this organelle elaborated the glycoprotein-rich acrosomic system. During steps 1-3, the Golgi apparatus produced, in addition to the proacrosomic granules, multivesicular bodies that became associated with the chromatoid body. NADPase was located within the four of five intermediate saccules of Golgi stacks, and TPPase was found in the last one or two saccules on the trans aspect of the stacks from steps 1 to 17 of spermiogenesis. CMPase was located within the thick saccular GERL elements found in the trans region of the Golgi apparatus from steps 1 to 7 of spermiogenesis, but the CMPase-positive GERL disappeared from the Golgi apparatus after its detachment from the acrosomic system at step 8. Th acrosomic system itself was reactive from CMPase and TPPase but was negative for NADPase, while the multivesicular bodies were CMPase and NADPase positive but unreactive for TPPase. Tritiated-fucose was readily incorporated within the Golgi apparatus of steps 1-17 spermatids; in steps 1-7 it was subsequently incorporated within the acrosomic system and multivesicular bodies. These various data indicated (1) that the Golgi apparatus of spermatids, although it loses its CMPase-positive GERL element in step 8, retains evidence of functional capacity until it degenerates in step 17; (2) that in early spermatids the various saccular components of the Golgi are specialized with respect to enzymatic activities; and (3) that each Golgi region may contribute in a coordinated fashion to the formation of the acrosomic system and multivesicular bodies.

Glycoprotein synthesis in Sertoli cells during the cycle of the seminiferous epithelium of adult rats: a radioautographic study.

Glycoprotein synthesis in Sertoli cells was investigated in rats sacrificed at various intervals (10, 30 min; 1, 4, 8, 24 h) after a single intratesticular injection of [3H] fucose. Thin sections of glutaraldehyde-fixed testes were radioautographed and the relative concentration of the label over the various cytoplasmic components of the Sertoli cells was analyzed quantitatively from electron microscopic photographs. The analysis was performed at the various stages of the cycle of the seminiferous epithelium grouped as follows: I-IV, V-VII, VIII-XI, XII-XIV. The incorporation of [3H] fucose into glycoproteins took place initially in the Golgi apparatus of the cells and the labeled glycoproteins later migrated to the lysosomes and plasma membrane. The corrected grain counts over the various cytoplasmic components of the Sertoli cell at various intervals after [3H] fucose injection, indicated that the synthesis of glycoproteins detected by radioautography after [3H] fucose injection was constant during the cycle of the seminiferous epithelium. Furthermore, the rate of transfer of the labeled glycoproteins from the Golgi apparatus to the lysosomes and plasma membrane was also noncyclic.

Phosphorylation of elk-1 by MEK/ERK pathway is necessary for c-fos gene activation during cardiac myocyte hypertrophy.

Cardiac hypertrophy is associated with specific alterations in myocardial gene expression; however, the exact mechanisms responsible for altered gene expression are poorly defined. The goal of this study was to investigate whether signaling kinases that are activated during cardiac hypertrophy directly modulate transcription factor activity and regulate gene expression. In an effort to understand this process, we focused our studies on the transcriptional activation of c-fos gene through the serum response element (SRE)/ternary complex factor (TCF) element, during phenylephrine-induced myocyte hypertrophy. In this study, we show that phosphorylated Elk-1, a TCF, binds to c-fos SRE and its binding to SRE is increased upon phenylephrine stimulation. Phenylephrine treatment activates phosphorylation of Elk-1 in the nucleus within five minutes and Elk-1-dependent transcriptional activation is abolished by inhibitors selective for MEK/ERK kinases. These studies implicate that phosphorylation of Elk-1 by ERK kinase pathway is important for early gene activation during phenylephrine-induced myocyte hypertrophy.

[Ca2+]i homeostasis and cyclic nucleotide relaxation in aorta of phospholamban-deficient mice.

Phospholamban (PLB), a protein localized in the sarcoplasmic reticulum (SR), inhibits the SR Ca2+-ATPase; phosphorylation of PLB relieves this inhibition. We previously reported significant differences in contractility in aorta from mice in which the gene for PLB was ablated (PLB-). In this study, we measured intracellular Ca2+ concentration ([Ca2+]i) with fura 2 in the intact mouse aorta to more directly test the hypothesis that these changes are ascribable to altered SR function in vivo. Ten micromoles per liter of the alpha-agonist phenylephrine (PE) increased [Ca2+]i monotonically to a steady state in the wild-type aorta. In contrast, in PLB- aorta there was an initial rapid increase to a peak [Ca2+]i, which then decreased to a steady state that was lower than that in the wild type. Upon removal of the stimulus (either PE or KCl), the decrease in [Ca2+]i was two times as fast in the PLB- as in the wild-type aorta. There were no significant differences between PLB- and wild-type aortas in the concentration vs. force relations or the time courses of relaxation in response to forskolin or sodium nitroprusside. Interestingly, stimulation of the cAMP pathway before cGMP pathway activation resulted in a significant increase in sensitivity and a difference in relaxation parameters between PLB- and wild-type aortas. Western blot analysis indicated that the PLB-to-sarcoendoplasmic reticulum Ca2+ATPase ratio in the mouse aorta was similar to that in the heart; 20-fold more aortic than heart homogenate was required to achieve a similar level of immunoreactivity. Our data indicate that PLB can play a major role in modulating smooth muscle [Ca(2+)](i) but only a minor role, if any, in cyclic nucleotide-mediated relaxation.

Enhanced Ca2+ channel currents in cardiac hypertrophy induced by activation of calcineurin-dependent pathway.

Cardiac-specific expression of an activated calcineurin protein in the hearts of transgenic (CLN) mice produces a profound hypertrophy that rapidly progresses to heart failure. While calcineurin is regulated by Ca2+, the potential effects of calcineurin on cardiac myocyte Ca2+ handling has not been evaluated. To this end, we examined L-type Ca2+ currents (I(Ca)) in left ventricular myocytes. CLN myocytes had larger (approximately 80%) cell capacitance and enhanced I(Ca) density (approximately 20%) compared with non-transgenic (NTG) littermates, but no change in the current-voltage relationship, single-channel conductance or protein levels of alpha 1 or beta 2 subunit of L-type Ca2+ channels. Interestingly, the kinetics of I(Ca) inactivation was faster (approximately two-fold) in CLN myocytes compared with NTG myocytes. Ryanodine application slowed the rate of I(Ca) inactivation in both groups and abolished the kinetic difference, suggesting that Ca2+ dependent inactivation is increased in CLN myocytes due to altered SR Ca2+ release. Treatment of CLN mice with Cyclosporine A (CsA), a calcineurin inhibitor, prevented myocyte hypertrophy and changes in I(Ca) activity and inactivation kinetics. However, there was no direct effect of CsA on I(Ca) in either NTG or CLN myocytes, suggesting that endogenous calcineurin activity does not directly regulate Ca2+ channel activity. This interpretation is consistent with the observation that I(Ca) density, inactivation kinetics and regulation by isoproterenol were normal in cardiac-specific transgenic mice expressing calcineurin inhibitory protein domains from either Cain or AKAP79. Taken together these data suggest that chronic activation of calcineurin is associated with myocyte hypertrophy and a secondary enhancement of intracellular Ca2+ handling that is tied to the hypertrophy response itself.