The effects of colony-stimulating factor-1 on the number and ultrastructure of osteoclasts in toothless (tl) rats and osteopetrotic (op) mice

The role of colony-stimulating factor-1 (CSF-1 or M-CSF) in osteoclast development is illustrated by observations that administration of exogenous CSF-1 increases osteoclast number and improves the skeletal sclerosis of two osteopetrotic mutations, toothless (tl) in the rat and osteopetrotic (op) in the mouse. We examined the effects of CSF-1 treatment on the number and ultrastructure of osteoclasts in the tibial metaphysis of normal and mutant animals of both stocks to understand the similarities and differences between these two mutations. Osteoclasts from normal animals of both stocks were abundant and possessed the ultrastructural features of active cells. These included apical areas in contact with mineralized surfaces with tightly apposed clear zones, extensive ruffled borders, and a vacuolated cytoplasm with numerous mitochondria. In toothless rats osteoclasts were difficult to locate and those present had poorly defined ruffled borders, fewer cytoplasmic vacuoles, and a basal membrane with both smooth and ruffled areas. Large lipid accumulations were often found near tl osteoclasts. Osteoclasts in op mice were difficult to find, but more numerous than in tl rats. Unlike tl osteoclasts, those of op mice possessed very well developed ruffled borders, small clear zones, and large electron-dense cytoplasmic inclusions. These cells also had unusual basal membranes with both smooth and ruffled regions. CSF-1 treatment increased the number of osteoclasts in both mutant stocks, normalizing the numbers in op mice, but not tl rats. CSF-1 injections caused dramatic changes in the morphology of tl osteoclasts, including increased incidence and size of ruffled borders and cytoplasmic vacuolization. The growth factor had little effect on ruffled borders or clear zones in op mice. Interestingly, mutant osteoclasts of both stocks exhibited a ruffled basal membrane in response to CSF-1 treatment. This increase in membrane ruffling may reflect the ability of CSF-1 to promote rapid formation of osteoclasts from mononuclear precursors in a more permissive microenvironment. Our data indicate that CSF-1 is not required for the development of at least some osteoclasts. The differences in response to CSF-1 treatment which we report lead us to speculate that additional factors may be involved in osteoclastogenesis.     

A defect in beta-galactosidase of B lymphocytes in the osteopetrotic (op/op) mouse

Tweezer-like receptor molecules have proven their potential for molecular recognition on several occasions. We decided to make twofold use of this receptor design: firstly to learn whether simple molecular forceps consisting of two peptide chains linked by a spacer are able to selectively bind to small peptides, and secondly to investigate the importance of structural preorganization for the characteristics of the receptors. We prepared two encoded combinatorial libraries based on this design, featuring two combinatorial tripeptide chains held by different scaffolds: the use of chenodeoxycholic acid as spacer provided a rigid scaffold for the forceps, whereas linking the peptide chains by a pentamethylene chain yielded a very flexible forceps structure. Molecules from the cholic acid library recognize and discriminate various enkephalins with micromolar affinities. Molecules from the flexible library show distinct interactions with the enkephalins as well, but the specificity and affinity are clearly diminished. Thus, although the interactions of molecular forceps with peptides are not crucially dependent on structural preorganization, receptors with a rigid design are clearly superior to flexible molecular forceps.     

Hypophosphatemia and the development of rickets in osteopetrotic (op/op) mice

Our previous work has shown that op/op mice hyperabsorb dietary calcium in the vitamin D-deficient state and shunt that calcium into bone. Under these conditions, the op/op mice are hypocalcemic. The purpose of this study was to examine calcium metabolism and bone mineralization in vitamin D-deficient op/op mice. First, the op/op mice and their normal littermates were placed on a vitamin D-deficient, low phosphorus diet to limit bone mineralization. Under these circumstances, op/op mice survived, even when calcium was also removed from the diet. If the diet contained phosphate, op/op mice died from hypocalcemic tetany when calcium was also removed from the diet. Furthermore, serum calcium levels became similar to wild type in the op/op mice administered the vitamin D-deficient, low phosphorus diet, and op/op mice were able to increase serum calcium in response to 1,25-dihydroxyvitamin D3. The op/op mice developed rickets when their serum phosphorus level was too low to support bone mineralization. The op/op mice became hypophosphatemic on regimens in which normal mice were able to maintain normal serum phosphorus levels. It appears that the op/op mouse simply requires a higher dietary calcium and phosphorus level to prevent rickets and hypocalcemic tetany since the bone is not available as a source of these minerals. However, the ability of the op/op mouse to mineralize bone at low serum calcium and phosphorus levels remains unexplained.     

Effect of macrophage colony-stimulating factor (M-CSF) on mouse immune responses in vivo

We examined the effects of recombinant human M-CSF (rhM-CSF) on mouse macrophages and immune responses in vivo. Intraperitoneal administration of rhM-CSF (20-500 microgram/ml) increased Mac-1+ cell numbers in the peritoneal cavity. The tumoricidal activities of the macrophages from vehicle-administered (V-M phi) and from rhM-CSF-administered (M-M phi) mice were the same as those observed in vitro. However, when activated by lipopolysaccharide (LPS), the tumoricidal activity of M-M phi was stronger than that of V-M phi. Intravenous administration of rhM-CSF (500 micrograms/gk) increased the number of spleen cells. Flow cytometric analysis showed that administration of rhM-CSF increased Mac-1+, B220+ and NK 1.1+ cell counts in the spleen. However, CD4+ and CD8+ cell numbers did not change. Concomitant increases were observed in levels of IL-4 and IL-10 in mouse serum following rhM-CSF administration, but no significant changes were observed in the serum level of IFN-gamma. In experiments involving mouse immune responses, the administration of rhM-CSF reduced the contact sensitivity (CS) reaction against picryl chloride (PC) and augmented IgE production in response to 2,4-dinitrophenyl (DNP), but did not affect the production of either IgM or IgC1. These results suggest that administration of rhM-CSF not only activates murine macrophages, but modulates antigen-specific immune responses in vivo.     

Anatomy and function of the reproductive tract in the captive male tree shrew (Tupaia belangeri)

The reproductive anatomy of the male tree shrew (Tupaia belangeri) was examined and compared with other Tupaiidae. The testes are located prepenially in a pigmented scrotum which is fused to the base of a pendulous penis. The terminal portion of the vas deferens is differentiated into an ampullary gland and joins the duct of the seminal vesicle to form a short ejaculatory duct. The prostate is a compact bilateral body drained by a main collecting duct. In the aggregate, these features indicate that the reproductive system in Tupaia is primate in character. Testicular function in tree shrews is affected by both social and seasonal factors. When males were housed communally, the majority exhibited testicular degeneration accompanied by a loss in the weight and fructose content of the seminal vesicles and in pigmentation of the scrotum. These changes may be due to the presence of dominant conspecifics since animals kept in isolation undergo normal sexual development. Animals captured throughout the year and isolated show seasonal fluctuations in androgenic and spermatogenic function. Reproductive capacity is maximal during the winter and minimal during the summer. Local environmental factors appear to regulate reproductive function so that the greatest number of births occur during the dry season.     

Location of a membrane-bound carbonic anhydrase isoenzyme (CA IV) in the human male reproductive tract

We studied the location of a membrane-bound carbonic anhydrase (CA IV) in the human male reproductive tract using a specific antiserum to human CA IV in conjunction with immunoblotting, immunoperoxidase, and immunofluorescence techniques. The microvilli and apical plasma membrane of the epithelial cells and the subepithelial smooth muscle layer of the epididymis, ductus deferens, and ampulla of the ductus deferens showed specific staining for CA IV. The epithelial cells of the prostate and seminal vesicle failed to stain for CA IV, however, whereas the subepithelial smooth muscle layer showed positive staining. No specific staining for CA II was seen in the epithelium of the epididymal duct or the proximal ductus deferens. The presence of CA IV in the epididymis was confirmed by immunoblotting, which revealed 35 KD and 33 KD polypeptides. The results show that the microvilli and the apical plasma membrane of the lining epithelium of the epididymal duct, ductus deferens, and ampulla of the ductus deferens contain the membrane-bound carbonic anhydrase isoenzyme IV. The presence of the enzyme in the epithelium of the epididymis and ductus deferens is probably linked to the acidification of the epididymal fluid that prevents premature sperm activation. Its physiological role in the smooth muscle cells remains to be elucidated.     

The effect of diabetes on sexual behavior and reproductive tract function in male rats

As part of a study of the diversity of myosins, we have cloned a cDNA encoding a myosin-like protein from Arabidopsis thaliana. This is the first molecular motor of any kind to be cloned from a higher plant. The predicted polypeptide (molecular weight 131 kDa) has a motor domain (head) very similar to those of other myosins, but the remainder of the sequence is unusual. The tail contains four potential calmodulin binding sites ("IQ-motifs"), but no sequence motifs suggestive of actin or phospholipid binding, like those found in other myosins. There is also a small region of probable alpha-helical coiled-coil, which suggests that the molecule could be dimeric, though unlikely to form filaments. The N-terminal and C-terminal regions of the molecule are unique. We present a phylogenetic analysis of myosin head sequences, which suggests that this is a new type of myosin.     

Correction of murine galactosialidosis by bone marrow-derived macrophages overexpressing human protective protein/cathepsin A under control of the colony-stimulating factor-1 receptor promoter

Galactosialidosis (GS) is a human neurodegenerative disease caused by a deficiency of lysosomal protective protein/cathepsin A (PPCA). The GS mouse model resembles the severe human condition, resulting in nephropathy, ataxia, and premature death. To rescue the disease phenotype, GS mice were transplanted with bone marrow from transgenic mice overexpressing human PPCA specifically in monocytes/macrophages under the control of the colony stimulating factor-1 receptor promoter. Transgenic macrophages infiltrated and resided in all organs and expressed PPCA at high levels. Correction occurred in hematopoietic tissues and nonhematopoietic organs, including the central nervous system. PPCA-expressing perivascular and leptomeningeal macrophages were detected throughout the brain of recipient mice, although some neuronal cells, such as Purkinje cells, continued to show storage and died. GS mice crossed into the transgenic background reflected the outcome of bone marrow-transplanted mice, but the course of neuronal degeneration was delayed in this model. These studies present definite evidence that macrophages alone can provide a source of corrective enzyme for visceral organs and may be beneficial for neuronal correction if expression levels are sufficient.     

Effect of cytochalasin B on the adhesion of mouse peritoneal macrophages

The adhesion of normal mouse macrophages to glass surfaces was reduced by nontoxic levels (1-50 mug/ml) of cytochalasin B in combination with a centrifugal force (1,000-8,000 g). Macrophages nonspecifically activated by Corynebacterium acnes were also detached by this treatment, but less effectively. The effects of cytochalasin B treatment on these cells were shown to be reversible. After detachment, the cells reattached to glass, appeared morphologically normal, and behaved like untreated cells as judged by adhesion, acid phosphatase levels, and phagocytosis. The effect of cytochalasin B on several parameters of phagocytosis by normal macrophages was also examined. The results demonstrate that cytochalasin B can be used to detach macrophages from surfaces and suggest a functional relationship between phagocytosis and macrophage adhesion to surfaces. Furthermore, the effect of cytochalasin B on adhesion of phagocytic cells provides a probe for further investigation of the adhesion of cells to surfaces.     

Distribution of Na,K-ATPase is normal in the inner ear of a mouse with a null mutation of the glucocorticoid receptor

This study was performed in order to test the hypothesis that the glucocorticoid hormone stimulates the formation of Na,K-ATPase in the inner ear of the mouse. An immunohistochemical study with respect to the presence and distribution of glucocorticoid receptors (GR) and Na,K-ATPase in the vestibular and cochlear regions of the inner ear was performed on a C57BL mouse with a null mutation of the glucocorticoid receptor (GR mutant mouse). The wild type C57BL mouse and the CBA mouse served as normal controls. As expected, the homozygous GR mutant mouse showed no specific staining for GR in the inner ear. The heterozygous GR mutant mouse showed faint staining of GR in the spiral limbus, the spiral ganglion, the organ of Corti and the utricle. This staining was markedly less than in the wild type C57BL mouse. Antibody labelling of Na,K-ATPase in the inner ear showed no significant difference between the homozygous and the heterozygous GR mutant mouse as compared to the control wild type C57BL mouse or the CBA mouse. Although earlier studies have shown a positive correlation between levels of glucocorticoid hormone in serum and the concentration of Na,K-ATPase in the inner ear, the hypothesis that glucocorticoid hormones alone stimulate the formation of Na,K-ATPase in the inner ear could not be confirmed by this study. Thus other regulating substances must be considered.     

MRL/lpr and MRL+/+ macrophage DNA synthesis in the absence and the presence of colony-stimulating factor-1 and granulocyte-macrophage colony-stimulating factor

Macrophage accumulation and proliferation as well as altered macrophage properties have been observed in autoimmune MRL mice. To determine whether there might be innate differences in the proliferative responses, we examined the DNA synthesis responses of peritoneal macrophages and macrophages derived in vitro from bone marrow precursors (bone marrow-derived macrophages (BMM)). Murine peritoneal exudate macrophages normally require the addition of macrophage CSF (CSF-1) to enter cell cycle in vitro. In contrast, we have found that many thioglycollate-induced adherent peritoneal macrophages, but not resident peritoneal macrophages, from both MRL/lpr and MRL+/+ mice atypically underwent DNA synthesis even in the absence of added CSF-1. They also responded very well to granulocyte-macrophage CSF. These findings may help to explain the appearance of increased macrophage numbers in MRL lesions. In contrast to a previous report, it was found that MRL/lpr and MRL+/+ BMM did not have an enhanced response to CSF-1 and that modulation of CSF-1 receptor expression was not more rapid in MRL BMM. We also found no evidence for abnormal CSF-1 internalization and degradation or for the lpr mutation to have any enhanced effect on BMM survival in the absence of CSF-1. TNF-alpha lowered the DNA synthesis response to CSF-1 of MRL/lpr BMM rather than enhanced it, as has been reported. Our data suggest that the enhanced accumulation of macrophages in the MRL/lpr kidney cannot be explained by a proposed model of enhanced responsiveness of MRL/lpr BMM to CSF-1, including a contribution by TNF-alpha.     

Effect of short photoperiodic cycles on male genital tract and testicular parameters in male goats (Capra hircus)

This study was performed in adult male goats in which seasonal variations were abolished by rapid alternations of long days and short days. These treatments have been shown previously to prevent seasonal changes in the hypothalamo-pituitary axis and to maintain testis weight and sperm production at a high level. The experimental groups were exposed for 3 years to an alternation of either a 1 month short (16 h dark; 8 h light) and 1 month long (16 L; 8 D) photoperiod (2 month cycle; n = 5) or of a 2 month short and 2 month long photoperiod (4 month cycle; n = 4). The control groups were maintained in natural photoperiodic conditions (45 degrees N) and goats were slaughtered in the non-breeding season (end of April RS; n = 5) at the same period as light-treated bucks, or in the breeding season (end of September BS; n = 6). The total weight of the testes, the length and mean diameter of the seminiferous tubules of light-treated goats were similar to those in the breeding season, and higher than those in the non-breeding season. The total number of A0 spermatogonia was increased by light treatments as compared to control goats in the breeding and non-breeding season. The daily production of A1 spermatogonia, leptonene primary spermatocytes and round spermatids in light-treated goats was maintained at the peak breeding season level. The intra-testicular concentration of testosterone, total volumes of intertubular tissue and of Leydig cells, and the number of Leydig cells per testis did not differ between groups. Although the mean cross-sectional area of Leydig cells in light-treated goats was similar to this area in non-breeding season goats, it was significantly lower than that of breeding season goats. In conclusion, the rapid alternation of short and long days allowed an increase in all the germ cells from the A0 spermatogonia onwards, which was responsible for the maintenance of high spermatogenetic activity of light-treated goats.     

A GLRA1 null mutation in recessive hyperekplexia challenges the functional role of glycine receptors

Dominant missense mutations in the human glycine receptor (GlyR) alpha 1 subunit gene (GLRA1) give rise to hereditary hyperekplexia. These mutations impair agonist affinities and change conductance states of expressed mutant channels, resulting in a partial loss of function. In a recessive case of hyperekplexia, we found a deletion of exons 1-6 of the GLRA1 gene. Born to consanguineous parents, the affected child is homozygous for this GLRA1(null) allele consistent with a complete loss of gene function. The child displayed exaggerated startle responses and pronounced head-retraction jerks reflecting a disinhibition of vestigial brain-stem reflexes. In contrast, proprio- and exteroceptive inhibition of muscle activity previously correlated to glycinergic mechanisms were not affected. This case demonstrates that, in contrast to the lethal effect of a null allele in the recessive mouse mutant oscillator (Glra1 spd-ot), the loss of the GlyR alpha 1 subunit is effectively compensated in man.     

Responsiveness of the adult male rat reproductive tract to 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure: Ah receptor and ARNT expression, CYP1A1 induction, and Ah receptor down-regulation

Cardiovascular diseases (CVD), principally coronary heart disease (CHD), stroke, and congestive heart failure, continue to be the leading cause of death claiming nearly 1,000,000 lives annually and accounting for more than 40% of the deaths in the United States (American Heart Association). While cardiovascular disease is often viewed as a problem of the elderly, 45% of heart attacks occur among individuals less than 65 years old. Moreover, CVD is the second leading cause of death for those 45 to 64 years of age and the third leading cause of death for those 25 to 44 years old. In economic terms, the annual direct and indirect costs of heart attack and stroke are approximately $259 billion or $492,444/second, in the United States alone. Thus, from a human and economic perspective, heart and vascular diseases are an enormous burden worthy of significant attention. This review is not intended to ignore the 50% decline in age-adjusted rates for heart attack and stroke events over the preceding three decades, as summarized by the recent report of the Joint National Committee on the Prevention, Detection, Evaluation and Treatment of High Blood Pressure (JNC VI). However, progress has halted as coronary heart disease and stroke mortality rates have reached a plateau in recent years and appear to be rising. Consequently, this is an excellent time for re-examining our approach to the prevention and treatment of CVD. In this article, readers will find an overview of the CVD prevention and treatment topics and not an in-depth analytical or epidemiological assessment of risk factors and outcomes. First, a macroscopic approach to reducing CVD is presented, followed by a discussion emphasizing the critical importance of public health and community-based programs in the effort to significantly reduce the burden of hypertension and related cardiovascular morbidity and mortality. Effective public health programs can play a pivotal role in raising awareness, and more importantly, in facilitating lifestyle change, entry and retention in the healthcare system, and compliance with non-pharmacological as well as drug therapy.     

Effect of macrophage colony-stimulating factor on mouse NK 1.1+ cell activity in vivo

The effect of recombinant human macrophage colony-stimulating factor (rhM-CSF) on NK 1.1+ cell activity in vivo and in vitro was studied. An intravenous injection of rhM-CSF increased the numbers of NK 1.1+ cells in mouse spleen and blood and augmented the clearance of Yac-1 cells in vivo. Using a magnetic cell sorter (MACS), we purified NK 1.1+ cells from vehicle-injected and rhM-CSF-injected mouse spleen cells. More than 95% of the collected cells were NK 1.1 antigen-positive. NK 1.1+ cells purified from rhM-CSF-injected mouse spleen cells exhibited (a) higher cytotoxic activity against Yac-1 cells, (b) higher proliferative responsiveness to interleukin (IL)-2 and (c) a greater production of interferon (IFN)-gamma in response to IL-2 and IL-12 compared to cells purified from vehicle-injected mouse spleen cells in vitro. These results suggest that the administration of rhM-CSF increases NK 1.1+ cell numbers and activates the cells in vivo.     

Human plasminogen activator inhibitor-1 (PAI-1) deficiency: characterization of a large kindred with a null mutation in the PAI-1 gene

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue- and urokinase-type plasminogen activators, is considered a critical regulator of the fibrinolytic system. We previously reported a child with abnormal bleeding and complete PAI-1 deficiency caused by a frame-shift mutation in exon 4 of the PAI-1 gene. The purpose of this study was to provide genetic and clinical data on the extended pedigree of the original proband to better define the phenotype associated with PAI-1 deficiency. Allele-specific oligonucleotide hybridization was used to genotype individuals, and serum PAI-1 antigen was measured by enzyme-linked immunosorbent assay. By this approach we have identified 19 individuals who are heterozygous for the PAI-1 null allele and 7 homozygous individuals with complete PAI-1 deficiency. Clinical manifestations of PAI-1 deficiency were restricted to abnormal bleeding, which was observed only after trauma or surgery in homozygous affected individuals. A spectrum of bleeding patterns was observed, including intracranial and joint bleeding after mild trauma, delayed surgical bleeding, severe menstrual bleeding, and frequent bruising. Fibrinolysis inhibitors, including epsilon-aminocaproic acid and tranexamic acid, were effective in treating and preventing bleeding episodes. Other than abnormal bleeding, no significant developmental or other abnormalities were observed in homozygous PAI-1-deficient individuals. Heterozygous PAI-1 deficiency was not associated with abnormal bleeding, even after trauma or surgery. These observations define the clinical spectrum of PAI-1 deficiency and provide additional evidence to support the hypothesis that the primary function of plasminogen activator inhibitor-1 in vivo is to regulate vascular fibrinolysis.     

Macrophage lineage cells in inflammation: characterization by colony-stimulating factor-1 (CSF-1) receptor (c-Fms), ER-MP58, and ER-MP20 (Ly-6C) expression

Isopentenyl diphosphate (IPP), which is produced from mevalonic acid or other nonmevalonic substrates, is the universal precursor of isoprenoids in nature. Despite the presence of several isoprenoid compounds in plastids, enzymes of the mevalonate pathway leading to IPP formation have never been isolated or identified to our knowledge. We now describe the characterization of two pepper (Capsicum annuum L.) cDNAs, CapTKT1 and CapTKT2, that encode transketolases having distinct and dedicated specificities. CapTKT1 is primarily involved in plastidial pentose phosphate and glycolytic cycle integration, whereas CapTKT2 initiates the synthesis of isoprenoids in plastids via the nonmevalonic acid pathway. From pyruvate and glyceraldehyde-3-phosphate, CapTKT2 catalyzes the formation of 1-deoxy-xylulose-5-phosphate, the IPP precursor. CapTKT1 is almost constitutively expressed during the chloroplast-to-chromoplast transition, whereas CapTKT2 is overexpressed during this period, probably to furnish the IPP necessary for increased carotenoid biosynthesis. Because deoxy-xylulose phosphate is shared by the plastid pathways of isoprenoid, thiamine (vitamin B1), and pyridoxine (vitamin B6) biosynthesis, our results may explain why albino phenotypes usually occur in thiamine-deficient plants.     

Potentiation of diethylstilbestrol-induced alterations in the female mouse reproductive tract by transforming growth factor-alpha transgene expression

Neonatal estrogen exposure causes numerous abnormalities in the female reproductive tract, including carcinogenesis. One mechanism by which neonatal estrogen elicits teratogenic and carcinogenic effects is epigenetic and involves the modulation of a number of estrogen-regulated genes including epidermal growth factor (EGF). Because of the evidence that there is an integral relationship between the EGF family, estrogen action, and the regulation of the growth and differentiation of the reproductive tract, we used transforming growth factor-alpha (TGF alpha) transgenic mice to investigate the interaction of constitutive TGF alpha expression with the potent estrogen diethylstilbestrol (DES) in the induction of reproductive-tract alterations. Our study was designed to determine whether TGF alpha expression could modulate DES-induced carcinogenesis of the female mouse reproductive tract. The animals were homozygous TGF alpha transgenic female mice from the MT42 line and the parental CD-1 outbred mice. The presence of the TGF alpha transgene significantly increased the incidence of DES-induced vaginal adenosis, uterine endometrial hyperplasia, uterine polyps, hypospadia, benign ovarian cysts, and pituitary adenomas. However, constitutive TGF alpha expression did not promote reproductive-tract neoplasia. This study demonstrates that TGF alpha participates in the regulation of developmental and morphogenic events in the Müllerian duct and urogenital sinus, suggesting a role for TGF alpha in the pathogenesis of reproductive-tract diseases. Furthermore, we showed that although constitutive expression of the TGF alpha transgene did have an effect on the reproductive tract, TGF alpha overexpression alone could not substitute for DES as a reproductive-tract carcinogen or as a promoter of uterine neoplasia, indicating that DES-induced carcinogenesis requires events in addition to the overexpression of this single peptide growth factor.