Globozoospermia in mice lacking the casein kinase II alpha' catalytic subunit.

Protein kinase casein kinase II (Ck2) is a cyclic-AMP and calcium-independent serine-threonine kinase that is composed of two catalytic subunits (alpha and alpha') and two regulatory beta-subunits. Ck2 is not a casein kinase in vivo, but over 100 substrates are known. The highly conserved amino acid sequences of its subunits and their broad expression suggest that Ck2 may have a fundamental role in cell function. Ck2 has been implicated in DNA replication, regulation of basal and inducible transcription, translation and control of metabolism. The Ck2alpha and Ck2alpha' isoforms (products of the genes Csnk2a1 and Csnk2a2, respectively) are highly homologous, but the reason for their redundancy and evolutionary conservation is unknown. We find here that Csnk2a2 is preferentially expressed in late stages of spermatogenesis, and male mice in which Csnk2a2 has been disrupted are infertile, with oligospermia and globozoospermia ('round-headed' spermatozoa). This is the first demonstration of a unique role for a Ck2 isoform in development. The primary spermatogenic defect in Csnk2a2-/- testis is a specific abnormality of anterior head shaping of elongating spermatids; this is the first defined gene that regulates sperm head morphogenesis. As the germ cells differentiate, they are capable of undergoing chromatin condensation, although many abnormal cells are deleted through apoptosis or Sertoli cell phagocytosis. The few that survive to populate the epididymis exhibit head abnormalities similar to those described in human globozoospermia, thus Csnk2a2 may be a candidate gene for these inherited syndromes.

The fine structure of the ventral intersegmental abdominal muscles of the insect Rhodnius prolixus during the molting cycle. I. Muscle structure at molting.

Rhodnius prolixus, a South American insect, molts five times in its development to an adult after emerging from the egg. Each molting cycle is triggered with a blood-meal. The ventral intersegmental abdominal muscles of Rhodnius develop during each molting cycle and are functional at molting. The fine structure of these fully developed muscles from fourth stage larval insects is studied. They have the characteristic structure of slow muscles. They have multiple motor nerve endings, and the myofibrils are poorly defined in cross-section. Longitudinal sections show long sarcomeres (8-10 micro), irregular Z-lines, and no apparent H zones. No M line is seen. Transverse sections through the A-band region show that each hexagonally arranged thick filament is surrounded by 12 thin filaments. Two thin filaments are shared by two neighboring thick filaments. The ratio of thin to thick filaments is 6:1. This structure is related to that found in vertebrate skeletal muscle and insect flight muscle.

The fine structure of the ventral intersegmental abdominal muscles of the insect Rhodnius prolixus during the molting cycle. II. Muscle changes in preparation for molting.

The development of the ventral intersegmental abdominal muscles of Rhodnius prolixus is triggered by feeding. The early muscle (1 day after feeding) contains essentially nonstriated fibrils. However, in cross-sections, areas indicating early I bands, Z lines, and A bands can be recognized. Interdigitating thick and thin myofilaments do not assemble into a precise lattice until sometime between 4 and 5 days after feeding. As development continues, the number of fibrils increases, the region corresponding to the Z line increases in density, and the fibrils contain more recognizable striations. The newly formed fibrils broaden as myofilaments are added peripherally. At all stages throughout development, the ratio of thin to thick myofilaments is always 6:1. The formation of fibrils in the abdominal muscles of Rhodnius is different from that in chick embryo skeletal muscle. The major differences are that at all stages in Rhodnius there are (1) a constant ratio of thin to thick myofilaments, and (2) detectable Z-line material. Other findings in Rhodnius suggest (1) that fusion of mononucleated cells with the multinucleated muscle cell occurs, (2) that microtubules develop in the tendon cell concomitantly with development of myofibrils in the associated muscle cell, and (3) that filaments 55A in diameter aggregate into microtubules.

Evidence that coated vesicles isolated from brain are calcium-sequestering organelles resembling sarcoplasmic reticulum.

Coated vesicles from the brain have been purified to near morphological homogeneity by a modification of the method of Pearse. These vesicles resemble sarcoplasmic reticulum fragments isolated from skeletal muscle. They contain proteins with 100,000- and 55,000-dalton mol wt which co-migrate on polyacrylamide gels, in the presence of sodium dodecyl sulfate, with the two major proteins of the sarcoplasmic reticulum fragment. These vesicles contain adenosine triphosphatase (ATPase) activity which is stimulated by calcium ions in the presence of Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.), displaying maximal activity at 8 x 10(-7) M Ca ++. They take up calcium ions from the medium, and this uptake is stimulated by ATP and by potassium oxalate, a calcium-trapping agent. The 100,000-dalton protein of the coated vesicles displays immunological reactivity with an antiserum directed against the 100,000-dalton, calcium-stimulated ATPase of the sarcoplasmic reticulum. As with the sarcoplasmic reticulum fragment, this protein becomes radiolabeled when coated vesicles are briefly incubated with gamma-labeled [32P]ATP. The possible functions of coated vesicles as calcium-sequestering organelles are discussed.

Effect of varying amounts of ascorbate on collagen, elastin and lysyl oxidase synthesis in aortic smooth muscle cell cultures.

In the presence of ascorbate, there is an increase in collagen synthesis with a concomitant decrease in insoluble elastin and lysyl oxidase activity in cultured rabbit aortic smooth muscle cells. While the addition of 0.5 micrograms ascorbate per ml of medium enhances collagen synthesis and accumulation, detectable insoluble elastin and lysyl oxidase activity remain essentially unchanged. However, at 2 micrograms ascorbate per ml, the integrity of the insoluble elastin is lost and lysyl oxidase activity is decreased. These studies suggest that by modifying the levels of ascorbate in the culture medium one can alter the nature of the extracellular matrix produced by the smooth muscle cells.

Alteration of the extracellular matrix of smooth muscle cells by ascorbate treatment.

The protein composition in the extracellular matrix of cultured neonatal rat aortic smooth muscle cells has been monitored over time in culture. The influence of ascorbate on insoluble elastin and collagen has been described. In the absence of ascorbate, the cells accumulate an insoluble elastin component which can account for as much as 50% of the total protein in the extracellular matrix. In the presence of ascorbate, the amount of insoluble collagen increases, while the insoluble elastin content is significantly less. When ascorbate conditions are varied at different times during the culture, the extracellular matrices are altered with respect to collagen and elastin ratios. The decrease in elastin accumulation in the presence of ascorbate may be explained by an overhydroxylation of tropoelastin. Approximately 1/3 of the prolyl residues in the soluble elastin fractions isolated from cultures grown in the presence of ascorbate are hydroxylated. Since the insoluble elastin accumulated in these cultures contain the unique lysine-derived cross-links in amounts comparable to aortic tissue, this culture system proves ideal for studying the influence of extracellular matrix elastin on cell growth and metabolism.

Long-term treatment of neonatal aortic smooth muscle cells with beta VLDL induces cholesterol accumulation.

A model for smooth muscle derived foam cells was developed by treating smooth muscle cells isolated from the aortae of neonatal rabbits with beta VLDL for up to 1 month. Hyperlipidemic beta VLDL isolated from cholesterol fed rabbits induced proliferation of the cells that were maintained in lipid deficient serum. In addition, the lipoprotein fraction stimulated [14C]oleic acid incorporation into [14C]cholesteryl ester, even in cultures that had been chronically exposed to the lipoprotein. The accumulation of cholesterol was evaluated and small amounts of cholesteryl ester were demonstrated in cultures treated for 3 days with beta VLDL. However, continued exposure to the lipoprotein resulted in larger elevations in total cholesterol, approximately 65% of which was in the esterified form in cultures treated with 100 micrograms beta VLDL/ml for 24 days. When cholesterol levels were examined as a function of time, it was determined that both total cholesterol and cholesteryl ester levels increased. Approximately 2-3 weeks after lipoprotein was introduced to the culture, maximum levels were attained. Triglyceride levels were also measured and found to increase more than two-fold in cultures that had been incubated in the presence of beta VLDL for 24 days, when compared to cultures incubated in its absence. Examination of the cultures by electron microscopy revealed intracytoplasmic lipid droplets in beta VLDL treated cells. These results suggest that beta VLDL treatment of neonatal aortic smooth muscle cells provides an ideal model in which to study the lipid laden smooth muscle cells that characterize the atherosclerotic plaque.

Detection of m2 muscarinic acetylcholine receptor mRNA in human corpus cavernosum by in-situ hybridization.

Relaxation and contraction of the smooth muscle of human corpus cavernosum (HCC) of the penis is essential for penile erection and detumescence. Relaxation of the smooth muscle is controlled, locally, by cholinergic, adrenergic and nonadrenergic, noncholinergic neurotransmitters as well as by the vascular endothelium, which lines the lacunar spaces and releases endothelium-derived relaxing factor. Cholinergic neurotransmitters are postulated to act on other neuroeffectors and on the endothelium rather than act directly on the trabecular smooth muscle. We use in-situ hybridization to determine the presence and distribution of muscarinic acetylcholine receptor (mAChR) subtype mRNA in HCC. First, we verified that the riboprobe used for in-situ hybridization is specific for m2 mAChR subtype using Chinese Hamster Ovary cells transfected with m2 mAChR subtype. Then, we validated that the tissue obtained from surgical specimens is adequate for in-situ hybridization. The data demonstrate that m2 mAChR subtype mRNA is expressed in HCC smooth muscle cells. m2 mAChR subtype mRNA expression is not detected in endothelium or nerves. It is possible that this receptor subtype plays a role in smooth muscle contraction.

Biochemical and histochemical analysis of the enzyme arylsulfatase in human lesions of endodontic origin.

Lesions of endodontic origin are areas of inflammatory response which occur as a result of untreated disease process within the root canal system. Lysosomal hydrolytic arylsulfatase A and B have been identified as major enzymes initiating and propagating bone loss by degrading chondroitin-4-sulfate. The purpose of this investigation was to examine human lesions of endodontic origin for the presence of arylsulfatase A and B. Fifteen periapical lesions were obtained at the time of periapical surgery. The lesions were analyzed for the presence of arylsulfatases using the spectrophotometer by monitoring the liberated 4-nitrocatechol at 515-nm wavelength. The same lesions were examined histochemically using the electron microscope. Five control samples from healthy periodontal ligament were evaluated in a similar manner. The results showed higher levels of arylsulfatase A in lesions than in control tissues, and marked activity of arylsulfatase B in lesions, whereas no activity of this enzyme was detected in the control specimen. Histochemically, all lesions showed positive staining for enzyme activity, whereas the controls were negative. These findings indicate that arylsulfatase A and B play a role in the pathogenesis of human lesions of endodontic origin.

The fine structure of developing locomotor muscles of the pelagic tunicate, cyclosalpa affinis (Thaliacea: Salpidae).

Salps are free-swimming tunicates whose peculiar life history renders them ideal for developmental studies. The solitary salp reproduces asexually by budding a stolon containing the complete developmental sequence of the aggregate generation. The ultrastructure of developing locomotor muscle of the aggregate generation of Cyclosalpa affinis was studied. The early muscle contains essentially non-striated myofibrils. However, in transverse sections, , areas indicating early I-band A-bands can be recognized. As development continues, the number of fibrils increases, the Z-line appear, and the fibrils contain more recognizable striations. The fully developed muscle has the caracteristic structure of striated muscle. Longitudinal sections show sarcomeres with irregular and discontinuous (perforated) Z-lines; H-zones are not apparent. No M-lines are seen. Throughout development, the ratio of thin to thick myofilaments is always 2:1, the ratio found in all vertebrate striated muscle. Other finding in C affinis suggest that: (1) multinucleated muscle cells are formed by the fusion of mononucleated cells, (2) membranes of adjacement mononucleated cells destined to fuse form myelin figures, and (3) these myelin figures become closely associated with mitochondria.

[Determination of immunoglobulins in the aged. Normal range and changes in pathological conditions]. / Il dosaggio delle immunoglobuline nell'anziano. Ambito di normalità e variazioni in situazioni patologiche.

Serum IgA, IgG and IgM values in 238 normal aged subjects were compared with those in 100 normal adults. Both male and female aged subjects displayed a significant rise in IgA and a significant fall in IgM, whereas IgG values were not markedly different. It was found that IgA increased and IgM decrease by an average of 12 mg % and 10 mg % (17 mg % in the aged) per decade respectively. Values were also determines in 597 aged hospital patients and related to the disease for which they were admitted. Increases in all three Igs were noted in sclerotic cardiopathy, chronic cerebrovascular insufficiency, acute broncopneumopathy (IgA increase only in chronic forms), and gastroduodenal ulcer. Diverticulosis of the colon and acute pancreatitis, however, were accompanied by elevated IgA values only. Increases were particularly marked in chronic liver disease, less so in diseases of the gallbladder. Neoplasia was usually accompanied by higher Ig levels.

A new role for the A2b adenosine receptor in regulating platelet function.

BACKGROUND: Activation of platelets is a critical component of atherothrombosis and plays a central role in the progression of unstable cardiovascular syndromes. Adenosine, acting through adenosine receptors, increases intracellular cAMP levels and inhibits platelet aggregation. The A2a adenosine receptor has already been recognized as a mediator of adenosine-dependent effects on platelet aggregation, and here we present a new role for the A2b adenosine receptor (A2bAR) in this process. METHODS AND RESULTS: As compared with platelets from wild-type controls, platelets derived from A2bAR knockout mice have significantly greater ADP receptor activation-induced aggregation. Although mouse megakaryocytes and platelets express low levels of the A2bAR transcript, this gene is highly upregulated following injury and systemic inflammation in vivo. Under these conditions, A2bAR-mediated inhibition of platelet aggregation significantly increases. Our studies also identify a novel mechanism by which the A2bAR could regulate platelet aggregation; namely, ablation of the A2bAR leads to upregulated expression of the P2Y1 ADP receptor, whereas A2bAR-mediated or direct elevation of cAMP has the opposite effect. Thus, the A2bAR regulates platelet function beyond mediating the immediate effect of adenosine on aggregation. CONCLUSIONS: Taken together, these investigations show for the first time that the platelet A2bAR is upregulated under stress in vivo, plays a significant role in regulating ADP receptor expression, and inhibits agonist-induced platelet aggregation.

The effects of testosterone deficiency on the structural integrity of the penile dorsal nerve in the rat.

Androgens play a vital role in erectile function and are known to have a neuroprotective role in the nervous system. This study investigated, in a rat model, the effects of testosterone deprivation and replacement on the morphology of the dorsal nerve of the rat penis at the light microscopy level. Two weeks after castration, male rats were infused with vehicle alone or 44 mug of testosterone for 2 weeks. Age-matched, sham-operated control animals were used for comparisons. Penile tissue samples were removed for histological analyses. The following parameters were assessed: (1) total myelin sheath thickness; (2) density of nerve fibers; and (3) axon cross-sectional area per nerve fiber. Castration resulted in a significant increase in axon cross-sectional area compared to that of the control and testosterone-treated animals (6.97+/-0.59 microm(2) per fiber in control animals to 14.32+/-0.44 microm(2) per fiber in castrated animals). Qualitatively, there were signs of nerve degeneration, particularly myelin sheath degeneration, in all sample groups. We did not observe statistically significant changes in myelin sheath thickness. There was a trend of reduced nerve density. Nerve degeneration was not quantified since this study was performed at the light microscopic level. This study suggests that testosterone has a neuroprotective role in the nerve fibers of the dorsal nerve and testosterone deficiency may lead to different forms of nerve degeneration resulting in anatomic alterations, thus contributing to erectile dysfunction.

Long-term neonatal rat aortic smooth muscle cell cultures: a model for the tunica media of a blood vessel.

Cultured neonatal rat aortic smooth muscle cells have been used to study the synthesis and accumulation of extracellular matrix components in many laboratories. These cells are capable of accumulating large amounts of insoluble elastin in the extracellular matrix and can be maintained in culture for long periods of time without subcultivation. This study examined the elastin and collagen contents of such cells in culture for 5, 21 and 43 weeks. The percent elastin and collagen observed in the 43-week cultures were strikingly similar to that seen in the intact neonatal rat aorta. It should be noted that the percent collagen varied significantly between 5 weeks and 43 weeks, whereas that for elastin remained relatively constant throughout the same time course. Histological examination demonstrated that the elastin fibers in the extracellular matrix of the cultures were arranged in a pattern similar to the elastic lamellae of the aortic tunica media. Data presented here suggest that these cells in culture mimic the donor tissue from which they were derived with respect to elastin and collagen content as well as elastic fiber arrangement, and possibly represent an organotypic culture of the medial layer of a blood vessel.

Elution and uncoating of Coxsackievirus B3 by isolated HeLa cell plasma membranes.

Plasma membranes isolated from HeLa cells on discontinuous sucrose gradients were assayed for their capacity to elute and uncoat coxsackievirus B3 at 37 C. Because the viral receptors are limited to the surface of HeLa cells, the addition of radioactively labeled virus to the cells prior to cell homogenization provided a useful marker for locating the plasma membranes during the fractionation procedure. Four bands were formed on the discontinuous sucrose gradients with approximately 70% or more of the membrane-associated viral label being recovered in the most dense bands, designated as bands 3 and 4. Bands 3 and 4 also possessed the plasma membrane marker enzymes, Na+, K+ adenosine triphosphatase and 5'-nucleotidase and revealed typical structures characteristic of plasma membranes as revealed by electron microscopy. Pelleted and washed membranes from band 3 both eluted and uncoated B3 32P-labeled virus, whereas membranes from band 4 eluted virus but failed to uncoat it. The membranes from band 4 were shown to inhibit the viral uncoating activity when mixed with membranes of band 3. Characteristically, unfractionated homogenates of cell membranes eluted but did not uncoat virus. The finding of a naturally occurring inhibitor of virus uncoating provides for the first time a way to distinguish between the membrane activities of virus elution and virus uncoating. The inhibitor remains to be characterized.

Formation of elastic fibers and elastin in rabbit aortic smooth muscle cell cultures.

Cultured rabbit aortic smooth muscle cells produce elastic fibers and elastin in their extracellular matrix. Morphological analyses of elastic fibers indicate that they consist of two distinct components which play a major role in elastic early fiber formation namely, the amorphous component and the "microfibrillar" component. During elastogenesis, the early fiber consists of small bundles of filaments. Later, clearly distinguishable small conglomerates of amorphous material are found distributed among the bundles of filaments. The mature elastic fibers have large conglomerates of amorphous material with the filaments present within the interstices. Long term cultures of these cells in the second passage continue to accumulate elastin. The crosslinking amino acid isomers desmosine and isodesmosine, which are unique to insoluble elastin, increase with time in culture. Twenty-four hour pulses with 14C-proline followed by measurements of 14C-hydroxyproline in the collagenase resistant protein of the cell layer show that the synthesis of insoluble elastin is constant up to the 14 week period studied from the time the amorphous material becomes evident ultrastructurally.

Cyclin D3 and megakaryocyte development: exploration of a transgenic phenotype.

The roles of cell cycle regulatory proteins in megakaryocyte development are poorly understood. We have previously demonstrated that cyclin D3 is expressed in megakaryocytes and is induced upon treatment with Mpl ligand. Transgenic mice in which cyclin D3 is overexpressed in the megakaryocytic lineage show features similar to in vivo Mpl ligand treatment, including increased megakaryocyte number and ploidy. Terminal maturation and platelet production are not enhanced, however, and transgenic megakaryocytes show a defect in demarcation membrane development. We have examined expression of the transcription factor nuclear factor (NF)-E2, known to be involved in cytoplasmic maturation and platelet fragmentation, in these transgenic mice and controls treated with Mpl ligand. Our findings demonstrate marked induction of NF-E2 mRNA in control megakaryocytes in response to Mpl ligand, but no NF-E2 increase in transgenic cells, potentially explaining the lack of platelet increase in these transgenic mice. Transgenic megakaryocytes treated with Mpl ligand display a limited increase in NF-E2. In response to literature reports of Mpl ligand-induced transient increases in p21Cip1/WAF1 mRNA in polyploidizing megakaryocytic cell lines, we have examined p21 transcript levels in both normal and transgenic megakaryocytes. In normal mouse spleen, only a small percentage of megakaryocytes express detectable levels of p21 mRNA, with the majority of these cells expressing at high intensity. p21 levels are not affected by treatment with Mpl ligand, while the frequency of expressing cells increases transiently. Transgenic megakaryocytes exposed to Mpl ligand also show an increased frequency of p21-positive cells, and stimulation with Mpl ligand resulted in a further increase in this frequency. The nature of this effect will require further investigation.