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PubMed1999

Heparan sulfate proteoglycan expression is induced during early erythroid differentiation of multipotent hematopoietic stem cells.

Heparan sulfate (HS) proteoglycans of bone marrow (BM) stromal cells and their extracellular matrix are important components of the microenvironment of hematopoietic tissues and are involved in the interaction of hematopoietic stem and stromal cells. Although previous studies have emphasized the role of HS proteoglycan synthesis by BM stromal cells, we have recently shown that the human hematopoietic progenitor cell line TF-1 also expressed an HS proteoglycan. Immunochemical, reverse transcriptase-polymerase chain reaction (RT-PCR), and Northern blot analysis of this HS proteoglycan showed that it was not related to the syndecan family of HS proteoglycans or to glypican. To answer the question of whether the expression of HS proteoglycans is associated with the differentiation state of hematopoietic progenitor cells, we have analyzed the proteoglycan synthesis of several murine and human hematopoietic progenitor cell lines. Proteoglycans were isolated from metabolically labeled cells and purified by several chromatographic steps. Isolation and characterization of proteoglycans from the cell lines HEL and ELM-D, which like TF-1 cells have an immature erythroid phenotype, showed that these cells synthesize the same HS proteoglycan, previously detected in TF-1 cells, as a major proteoglycan. In contrast, cell lines of the myeloid lineage, like the myeloblastic/promyelocytic cell lines B1 and B2, do not express HS proteoglycans. Taken together, our data strongly suggest that expression of this HS proteoglycan in hematopoietic progenitor cell lines is associated with the erythroid lineage. To prove this association we have analyzed the proteoglycan expression in the nonleukemic multipotent stem cell line FDCP-Mix-A4 after induction of erythroid or granulocytic differentiation. Our data show that HS proteoglycan expression is induced during early erythroid differentiation of multipotent hematopoietic stem cells. In contrast, during granulocytic differentiation, no expression of HS proteoglycans was observed.

Cell Differentiation↗

PubMed2000

Characteristic features of the genotype and phenotype of hereditary spherocytosis in the Japanese population.

Hereditary spherocytosis (HS) is the most common hemolytic anemia of congenital origin in the Japanese population. Among 844 cases of 520 kindred with congenital red cell membrane disorders studied at the Kawasaki Medical School in the last 25 years (1975-1999), 407 cases (48.2%) of 215 kindred had HS. Among the recent 60 kindred with HS, autosomal dominant (AD) transmission was proven in 19. The remaining 41 non-AD HS included 1) homozygous patients with autosomal recessive inheritance, 2) HS patients with de novo gene mutations, and 3) mild HS with AD inheritance. The extent of clinical severity in the non-AD HS cases was nearly identical to that in the AD cases. The incidence of membrane protein abnormalities in our 60 Japanese HS kindred was unique: there were lower ankyrin deficiencies (7%), moderate band 3 deficiencies (20%), and much higher protein 4.2 deficiencies (45%), with 28% of unknown etiology. The incidence of membrane protein deficiencies corresponded to that determined by gene analyses; i.e., mutations mostly in band 3 and/or in protein 4.2 genes and fewer ankyrin gene mutations. In the band 3 gene, 11 mutations pathognomonic for HS were identified (3 frameshift and 8 missense mutations). There were 5 mutations of the protein 4.2 gene (3 missense mutations, 1 nonsense mutation, and 1 splicing mutation) pathognomonic for HS. On the other hand, 2 missense mutations were detected in the ankyrin gene in this study. The genetic abnormalities in our HS patients correlated well with the phenotypic ultrastructural abnormalities of red cell membranes in situ. Ankyrin mutations (ankyrin Marburg and ankyrin Stuttgart with frameshift mutations) were associated mostly with a disrupted cytoskeletal network, and band 3 mutations (band 3 Kagoshima with frameshift mutation) typically demonstrated anomalies of intramembrane particles (IMPs). Protein 4.2 mutations (homozygotes of protein 4.2 Nippon) with complete protein 4.2 deficiency showed abnormalities of both the cytoskeletal network and IMPs.

Animals↗

PubMed2000

Interleukin (IL)1beta, IL-1alpha, and IL-1 receptor antagonist gene polymorphisms in patients with temporal lobe epilepsy.

Proinflammatory cytokines, including interleukin (IL)-1beta, are known to modulate effects of neurotoxic neurotransmitters discharged during excitation or inflammation in the central nervous system (CNS). They also regulate development of glial scars at sites of CNS injury. To elucidate a genetic predisposition of temporal lobe epilepsy with hippocampal sclerosis (TLE-HS+), we studied polymorphisms in the IL-1beta, IL-1alpha, and IL-1 receptor antagonist (IL-1RA) genes in 50 patients with TLE-HS+ and in 112 controls. Fifty-three patients who had TLE without HS were also examined (TLE-HS-) as disease controls. The distribution of the biallelic polymorphism in the promoter region at position -511 of the IL-1beta gene (IL-1B-511) was significantly different both between TLE-HS+ patients and controls and between TLE-HS+ and TLE-HS- patients. The differences were due to overrepresentation of the homozygotes for IL-1B-511*2, which is suggested to be a high producer of IL-1beta, in TLE-HS+ patients compared with both controls and TLE-HS- patients. In contrast, there was no difference between TLE-HS- patients and controls. Our data suggest that, in the homozygotes for IL-IB-511*2, minor events in development such as febrile convulsions could set up a cascade leading to HS.

Adult↗

PubMed1992

Heat shock. A new approach for myocardial preservation in cardiac surgery.

BACKGROUND: Recent studies have indicated that heat shock (HS) induces protective genes and HS protein (HSP) expression, which enhances cellular tolerance to ischemic injury. The present study sought to determine if 42 degrees C blood cardioplegia could be used to induce HSP expression and improved myocardial salvage after 2 hours of cardioplegic arrest. METHODS AND RESULTS: To study this, pig hearts (n = 6) on cardiopulmonary bypass were subjected to HS by continuous infusion to the globally arrested heart of warm blood cardioplegia (K+ = 30 meq/l) at 42 degrees C for 15 minutes followed by 2 hours of intermittent hypothermic (4-6 degrees C) hyperkalemic (30 meq/l) crystalloid cardioplegic arrest and 1 hour of reperfusion (heat shock group). The control group (n = 6) was subjected to only 2 hours of hypothermic crystalloid cardioplegic arrest followed by 1 hour of reperfusion without HS. Left ventricular performance, coronary blood flow, and creatine kinase release were determined before arrest and during reperfusion. Prearrest control biopsies were taken in a separate group of pigs (n = 6) for both HSP and superoxide dismutase activity. Additional biopsies were taken for the same measurements in both control and HS groups at the completion of reperfusion. In a single additional pig in both the control and HS groups, biopsies were taken during the study to estimate changes in HSP expression. The cytosolic protein of ventricular tissue was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the HSP70 family protein was examined by Western blot analysis using monoclonal antibodies. HSP was found to be increasingly expressed from control levels after 15 minutes of HS pretreatment, increasing progressively to a level significantly above the non-HS group. Associated with this increased expression of HSP was a significant increase in superoxide dismutase activity in the HS animals and significant improvement in both global and regional functions and reduced creatine kinase release. CONCLUSIONS: The results show that preconditioning the heart with HS improves postischemic ventricular performance and attenuates cellular injury. HS induces a change in cellular metabolism, prompting the expression of HSP and improving antioxidant activity, leading to improved function and reduced tissue injury during ischemia and reperfusion.

Animals↗

PubMed1992

Binding of primitive hematopoietic progenitor cells to marrow stromal cells involves heparan sulfate.

Blast colony-forming cells (BI-CFC) and pre-colony-forming unit-granulocyte, monocyte (CFU-GM) in human bone marrow bind to marrow-derived stromal layers grown in the presence of methylprednisolone (MP+), but do not bind to stroma grown without MP (MP-). The BI-CFC bind to stroma and form colonies when overlaid with agar; the pre-CFU-GM bind to stroma and release CFU-GM into the supernatant culture medium (delta assay). These two classes of progenitor may represent similar stages of hematopoietic cell development. Their binding to stroma depends on the presence of heparan sulfate proteoglycan (HS-PG) in the extracellular matrix secreted by the stromal cells. Here, we have analyzed the functional and biochemical properties of HS-PG isolated from MP+ and MP- stromal cultures. HS-PG or isolated HS glycosaminoglycan (GAG) side chains partially blocked progenitor cell binding when they were added to the 2-hour binding phase of the BI-CFC or delta assays. Gel electrophoresis of HS-PG resolved more bands in matrix preparations from MP+ cultures than in preparations from MP- cultures. The blocking activity of the eluted MP+ HS-PG bands depended partly on the amount of GAG attached to the protein core and presumably partly on the structure of the core itself. Time course studies demonstrated that the HS-dependent phase of the binding interaction was limited to the first 30 to 60 minutes of the 2-hour binding phase. The different blocking effects of MP+ and MP- HS indicate that they have different biochemical properties. The HS-GAG in MP+ stroma has a higher degree of sulfation and a greater negative charge to mass ratio compared with MP- HS-GAG. Variations in HS may determine specific binding by hematopoietic progenitor cells and a heparan sulfate receptor is envisaged as acting in concert with further cell adhesion molecules (CAMs) on the progenitor cell surface.

Adult↗

PubMedDate not supplied

[Leptine participation in the development of liver steatosis and biliar lithiasis].

Obesity increases significantly the risk of developing several common gastrointestinal diseases such as gallstone disease (GD) and hepatic steatosis (HS). Elsewhere we have shown a relationship between HDL cholesterol, cholesterol saturation index, and leptin in obese patients loosing weight. Furthermore, leptin plays an important role facilitating HS and possibly in the associated inflammatory process. The aim of this study was to investigate the relationship between GD and HS. The sample was comprised by patients attending the unit for check-up. Subjects with visible stones or HS by ultrasound (cases) were compared with healthy controls. Demographic and body mass index (BMI) were recorded. Plasma leptin, insulin and serum lipids and lipoproteins levels were measured by standard methods. A total of 317 subjects were included in this study. They were divided in four groups as follows: GD (n=100), HS (n=84), GD + HS (n=33) and controls (n=100). The control group was significantly older (GD, 52.6+/-11.6; HS, 49.8+/-11.1; GD +HS, 51.6+/-10.5; 57.1+/-7.4), p< 0.05. BMI was higher in the HS groups (28.7 +/- 2.8) and GD +EH (29.0 +/- 3.8) than in the GD (27.4 +/- 4.3) and control (27.0 +/- 3.1) group, p<0.05. The GD group displayed the highest leptin levels (13.7 241 8.1), P < 0.05, whereas insulin levels were similar in all groups. Since GD and HS subjects have high plasma leptin levels compared with controls, our results suggest that leptin plays an important role in the pathophysiology of GD and HS.

Fatty Liver↗

PubMed2006

[Predictive value of fibrinogen and high-sensitivity C-reaction protein for cardiovascular events in patients with stable coronary artery disease].

OBJECTIVE: To investigate the predictive value of elevated fibrinogen and high-sensitivity C-reaction protein (hs-CRP) level on cardiovascular events in patients with stable coronary artery disease (CAD). METHODS: From January 2002 to November 2002, 185 patients (aged 47 - 85 years) with stable CAD referred for coronary angiography were enrolled and divided into control-F (fibrinogen level < or = 4.0 g/L, n = 104) and elevated-F (fibrinogen level > 4.0 g/L, n = 81), or control-hs (hs-CRP < or = 3.0 mg/L, n = 99) and elevated-hs (hs-CRP> 3.0 mg/L, n = 86). Exclusion criteria included cardiomyopathy, New York Heart Association class IV congestive heart failure, recent myocardial infarction or coronary artery revascularization and cancer. During three years follow-up, cardiovascular death, myocardial infarction, congestive heart failure, stroke and other vascular events were assessed. RESULTS: A total of 21 cardiovascular nonfatal events and 10 cardiovascular deaths were observed. Cardiovascular events was significantly higher in patients in elevated-F group than that in control-F group [23.46% vs. 11.54%, cholesterol-, body mass index-, smoking-, and hypertension-adjusted relative risk 1.97, 95% CI (1.68 to 2.40), P < 0.05] and in elevated-hs group than in control-hs group [24.42% vs. 10.10%, adjusted relative risk 2.32, 95% CI (1.76 to 2.89), P < 0.05]. The relative risk of cardiovascular events for patients with fibrinogen > 4.0 g/L and hs-CRP > 3.0 mg/L was 3.84 (P < 0.05), 95% CI (2.80 to 4.99) compared with patients with fibrinogen < or = 4.0 g/L and hs-CRP < or = 3.0 mg/L. CONCLUSION: Both fibrinogen and hs-CRP are independent important predictors of cardiovascular nonfatal and fatal events in patients with stable CAD. Combination of elevated fibrinogen and hs-CRP increased their predictive value for cardiac events.

Aged↗

PubMed2005

Heat shock protein 72 protects retinal ganglion cells in rat model of acute glaucoma.

PURPOSE: To investigate whether the induction of heat shock protein (HSP)72 by heat stress (HS) or zinc (Zn2+ ) administration can increase survival of retinal ganglion cells (RGC)in rat model of acute experimental glaucoma. METHODS: Acute glaucoma model was made by intracameral irrigation with BSS at 10(2) mmHg for two hours in right eyes of male Wistar rats. Glaucoma model rats were treated with HS once a week (six rats) or intraperitoneal injection of zinc sulfate (24.6 mg/kg) every two weeks (six rats), and were referred to as HS group and zinc group, respectively. Untreated model rats served as damage group (six rats). In control groups, quercetin (400 mg/kg) was intraperitoneally injected to inhibit the induction of heat shock proteins 6 hours before HS or zinc administration, and were referred to as HS+que group (six rats) and zinc+que group (six rats), respectively. Subsequent to 16 days of IOP elevation, the rats were sacrificed. Eyes were quickly enucleated, and the retinas were dissected. RGC were labeled with Nissl staining and counted under microscope. RESULTS: The average RGC density in normal Wistar rats was (2504+/-181) cells/mm(2). In damage group, it decreased to (2015+/-1111) cells/mm(2). The RGC densities at 1, 2, and 3 mm from the center of the optic nerve head were (2716+/-215), (2496+/-168), and (2317+/-171) cells/mm(2), respectively, for normal rats and (2211+/-133), (1969+/-154), and (1872+/-68) cells/mm(2), respectively, for damage group. The latter was significantly lower at all locations compared with the former (P=0.027 for each, Mann-Whitney test). The average RGC densities were (2207+/-200) cells/mm(2) for HS group, (2272+/-155) cells/mm(2) for zinc group, (1964+/-188) cells/mm(2) for HS+que group, (2051+/-214) cells/mm(2 )for zinc+que group and (2015+/-111) cells/mm(2) for damage group. There were significant differences in density of labeled RGCs among the five groups (P=0.040, Kruskal-Wallis test). Both HS and zinc group had higher RGC densities than damage group (P =0.036 between HS and damage group, P=0.019 between zinc and damage group, Mann-Whitney test). There was no significant difference in RGC density between control groups and damage group(P=-0.260 between HS+que and damage group, P-0.748 between zinc+que and damage group, Mann-Whitney test). CONCLUSIONS: The results demonstrated that the induction of HSP72 in RGCs by HS or zinc administration plays an important role in the survival of RGCs in rat model of acute glaucoma. A novel therapeutic approach to glaucoma through an enhanced induction of endogenous HSP72 could be possible.

Animals↗

PubMed1990

Early accumulation of heparan sulfate in neurons and in the beta-amyloid protein-containing lesions of Alzheimer's disease and Down's syndrome.

A monoclonal antibody (HK-249) that recognizes a glucosamine sulfate alpha 1----4 glucuronic acid-containing determinant in heparan sulfate (HS) chains of a basement membrane-derived heparan sulfate proteoglycan identified and immunolocalized HS specifically to the amyloid deposits in neuritic plaques (NPs), congophilic angiopathy (CA), as well as in neurofibrillary tangles (NFTs) and non-tangle-bearing neurons in the brains of Alzheimer's and Down's syndrome (DS) patients. Ultrastructural immunohistochemistry demonstrated that HS within neurons of Alzheimer's disease (AD) brain was localized to lipofuscin granules, an aging pigment previously shown also to contain beta-amyloid protein (BAP). Heparan sulfate also was localized to neurite-containing, nonfibrillar 'primitive' plaques that also demonstrated positive BAP immunoreactivity in both AD and DS brains. Antibodies to laminin, fibronectin, and a chondroitin sulfate proteoglycan failed to show positive immunostaining of the HS-containing sites described above. Analysis of DS patients at different ages revealed that HS accumulated within neurons of the hippocampus and amygdala as early as 1 day after birth. Young age-matched controls did not demonstrate similar positive HS immunoreactivity in neurons, whereas positive immunostaining for HS was observed in other regions thought to normally contain HS. The earliest deposition of BAP was first observed as 'amorphous' or 'diffuse' cortical deposits in DS brain in patients aged 18 and 24 years before the accumulation of fibrillar amyloid (observed in DS patients who are 35 years and older). These cortical deposits also contained positive HS immunoreactivity, implying that HS accumulation in conjunction with the BAP is an early event that ultimately may contribute to the early age-related accumulation (ie, as early as 35 years of age in DS) of NPs, NFTs, and/or CA. Furthermore the colocalization of HS and BAP in a number of specific locales in AD and DS brain indicates a possible interaction between these two macromolecules that may be important in lesion development in these two diseases.

Adolescent↗

PubMed1990

Influence of 7 days of hindlimb suspension and intermittent weight support on rat muscle mechanical properties.

Unloading the rat hindlimb results in a decrease in mass, especially in those muscles that normally have a load-bearing function. The present study was designed to evaluate the effect of intermittent periods of weight support in ameliorating this atrophic response. Adult male Sprague-Dawley rats were assigned to either a control (CON), a hindlimb suspended (HS), or a hindlimb suspended plus intermittent weight support (HS-WS) group. HS-WS rats were walked slowly on a treadmill at 0.2 m/s and a 19% incline for 10 min, every 6 h. After 7 d, the in situ mechanical properties of the soleus (Sol) and medial gastrocnemius (MG) were studied. Body weights of HS and HS-WS rats were 9 and 13% lower than CON. The SOl weight relative to body weight was 21 and 9% lower in HS and HS-WS than CON. Maximum tetanic tension relative to muscle mass was significantly lower in HS than CON, whereas HS-WS had values similar to CON. The MG weight relative to body weight was significantly lower in both suspended groups. The maximum tetanic tension relative to muscle weight was significantly elevated in HS-WS compared to CON, suggesting that weight support may have preferentially maintained the contractile protein component of the muscle. Contraction times were 25% faster (p less than 0.05) in the Sol and unchanged in teh MG of HS rats. For each muscle, the fatigue properties were similar in all groups. These data indicate that a low-force, short-duration exercise regime results in a significant functional recovery in the "slow" Sol, whereas the "fast" MG is less affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Airway responsiveness to adenosine 5' monophosphate following inhalation of hypertonic saline.

We wished to determine whether the refractory period after hypertonic saline (HS) challenge is due to mast cell mediator depletion. Therefore, the airway responsiveness to adenosine 5' monophosphate (AMP), which induces bronchoconstriction via mast cell histamine release, was determined after the inhalation of HS aerosol. Nine asthmatic subjects attended the laboratory on three occasions. On day 1 HS challenge was performed followed one hour later by a second HS challenge. On day 2 an AMP challenge was performed. On day 3 an HS challenge was performed followed one hour later by an AMP challenge. Airway responsiveness (PD35 sGaw) to an initial HS challenge ranged from 12 to 315 l of aerosol (mean 47 l). Airway responsiveness to a second HS challenge ranged from 8 to 800 l (mean 102 l p = 0.035, n = 9). Airway responsiveness to AMP increased from 0.44 to 14.0 mumol (mean 2.37 mumol) at baseline to 0.3 to 15.5 (mean 1.3 mumol) (p = 0.05) after HS challenge. There was a linear correlation between baseline AMP responsiveness and baseline HS responsiveness (r = 0.911, p = 0.001). There was no correlation between the degree of refractoriness and the change in AMP responsiveness (r = 0.1, p = 0.9). Thus airway responsiveness to AMP increased significantly after inhalation of HS aerosol and this increase was independent of refractory behaviour. Our results suggest that the refractory period to HS is not due to mediator depletion.

Adenosine Monophosphate↗

PubMed1989

[Pharmacokinetic interaction of ketoconazole, isoniazid and rifampicin].

Eight male tuberculous patients, between 20 and 60 years of age, were given Isoniazid 5 mg/kg and Ketoconazole 200 mg, first one at a time and then associated. Plasma concentrations were measured 0, 2 and 5 hs after taking the drugs. Isoniazid was measured by spectrophotometry and Ketoconazole by the microbiologic method with Candida albicans as test microorganism. When both drugs were given simultaneously Ketoconazole plasma concentration decreased 75% at 2 hs (p less than 0.025) and 85% at 5 hs (p less than 0.05), whereas that of Isoniazid remained unchanged (Table 1). Mean half-life of Isoniazid was 3.9 +/- 1.4 hs in 7 slow acetylators and 1.1 hs in one fast acetylator when given one at a time and 4.4 +/- 1.5 hs when given simultaneously. A similar study was conducted on 11 tuberculous patients who were given Rifampicin 10 mg/kg and Ketoconazole 200 mg, one at a time and concurrently. Rifampicin was measured by high pressure liquid chromatography. When Rifampicin and Ketoconazole were given concurrently plasma concentration of both drugs was reduced: Ketoconazole decreased 85% at 2 hs (p less than 0.025) and 98% at 5 hs (p less than 0.025) whereas Rifampicin decreased 45% at 2 hs (p less than 0.005) and 40% at 5 hs (p less than 0.005) (Table 2). Mean half-life of Rifampicin was 3.5 +/- 0.8 and 4.2 +/- 1.1 hs, respectively, when it was given alone and concurrently. Studies on chemical interactions between Isoniazid and Ketoconazole and between Rifampicin and Ketoconazole yielded negative results.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1989

Exercise effects on the size and metabolic properties of soleus fibers in hindlimb-suspended rats.

The soleus atrophies rapidly when a rat is subjected to hindlimb suspension (HS), probably as a result of a decrease in the force encountered by the muscle. To test this premise, adult female rats were HS and half the rats were exercised (HS-EX) on a treadmill for 1.5 h.d-1 at 20 m.min-1 and a 30% grade. After 4 weeks, the midbelly of the soleus was prepared for histochemical analysis. Fibers were typed as dark or light staining for myosin ATPase, alkaline preincubation. Fiber size and quantitative histochemical enzyme activities of succinate dehydrogenase (SDH) were determined using a computer enhanced image processing system. In comparison to age-matched controls, the soleus wet weight was 69 and 30% smaller in HS and HS-EX rats. The mean cross sectional area of the dark ATPase fibers was reduced by 46 and 18% and light ATPase fibers by 69 and 48% in the HS and HS-EX, respectively. The percent dark ATPase fibers increased from 10% in the control rats to 19 and 17% in the HS and HS-EX. In both suspended groups, SDH activities in light ATPase fibers were 40% higher than control. The SDH activity of the dark ATPase fibers of HS-EX was 20% higher than control, while the dark ATPase fibers of HS were similar to control. To determine the degree to which these increases in SDH could be related to reductions in fiber size rather than increases in the actual amount of protein, integrated activity (activity/min x area) was calculated per fiber.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

A cytoskeleton-associated plasma membrane heparan sulfate proteoglycan in Schwann cells.

Schwann cells cocultured with sensory neurons in a serum-free medium accumulate a single species of radiolabeled heparan sulfate proteoglycan (HS-PG) during incubation in medium containing 35SO4. This HS-PG was poorly extracted from cultures by solutions containing 1% Triton X-100 in low salt buffer or by solutions containing 1 M KCl, 4 M urea plus dithiothreitol, 1 mM Tris-HCl, 5 mM EDTA, or 100 micrograms/ml of heparin. The HS-PG was efficiently extracted, however, by 1% Triton X-100 in the presence of 1 M KCl or by 1% deoxycholate. These treatments solubilize both cell membranes and the Schwann cell cytoskeleton. In intact cells the HS-PG was digested by trypsin, indicating it was at least partially exposed on the cell surface. When solubilized HS-PG was applied to a column of octyl-sepharose CL-4B, more than 90% was retained by the column, but was quantitatively eluted by a solution containing 1% Triton X-100. In addition, the solubilized HS-PG could be incorporated into artificial phospholipid vesicles. These results indicate the HS-PG is an integral plasma membrane protein. The inability of low ionic strength solutions containing Triton X-100 to solubilize the HS-PG suggested it was bound to an additional structure. To determine whether the HS-PG was associated with the cytoskeleton we isolated cytoskeletons by detergent lysis of cells and centrifugation. The major protein components of isolated cytoskeletons were spectrin (Mr 225,000), vimentin (Mr 58,000), and actin (Mr 45,000). When 35SO4-labeled cells were used to prepare cytoskeletons approximately 80% of the total HS-PG was recovered in the cytoskeleton fraction. These results suggest the HS-PG is an externally exposed integral plasma membrane protein that is anchored to the Schwann cell cytoskeleton.

Animals↗

PubMed1986

Partial characterization of heparan and dermatan sulfate proteoglycans synthesized by normal rat glomeruli.

Rat glomerular heparan sulfate (HS) and dermatan sulfate (DS) proteoglycan synthesis was studied in vitro and in vivo. Incorporation of [35S]sulfate into macromolecules was linear over 16 h in vitro, and DS was the predominant glycosaminoglycan (GAG), while HS dominated in vivo incubations. Proteoglycans were found in the bottom 2/5 (high density) CsCl gradient fractions and eluted as two overlapping peaks from DEAE-Sephacel columns. The proportion of low density 35S-glycoproteins and 35S-proteoglycans increased with time. Two high buoyant density HS proteoglycans were extracted from glomeruli and eluted in DEAE peak I. The first, HS-tIA, had an Mr of 130 X 10(3) with Mr 12.5 X 10(3) GAG chains. This proteoglycan was released from the tissue by trypsin and was partially displaced by heparin treatment. In addition, it was rapidly released into the medium of label-chase experiments after which it migrated slightly more rapidly than HS-tIA in gels, with HS chains similar in length to its tissue counterpart. The second, HS-tIB, had an Mr of 8.6 X 10(3) with little or no attached protein. This proteoglycan was characterized as intracellular as it resisted release by trypsin treatment or heparin extraction in medium and was not detected in the medium of label-chase experiments. Two tissue DS proteoglycans were characterized. The first, DS-tIA, co-purified with HS-tIA and was the predominant proteoglycan synthesized during 4-h in vitro incubations. Like HS-tIA, it was rapidly released into medium and displaced from cell surfaces or tissue "receptors" by heparin or trypsin treatments. A second, Sepharose CL-6B-excluded DS proteoglycan from DEAE peak II, DS-tII, accumulated in tissue over 16 h in vitro. This proteoglycan was self-associating and contained clusters of iduronic acid residues along its Mr 26 X 10(3) DS chains. It resisted extraction from the tissue with heparin, trypsin, and detergent. No DS-tII was detected in the incubation medium. Instead, medium proteoglycans eluted as single Sepharose CL-6B-included peaks. DS chains from medium proteoglycans were shorter (Mr 18 X 10(3)) and had more regularly spaced iduronic acid residues than GAGs from DS-tII. The length and sulfation patterns of DS-mII GAG were similar to GAG from DS-tIA. Thus, glomeruli rapidly synthesized and released Sepharose CL-6B-included heparin-displaceable DS and HS proteoglycans while retaining a Sepharose CL-6B-excluded self-associating DS proteoglycan and an intracellular HS.

Animals↗

PubMed1995

Hormonal and cell density regulation of hepatic gamma-glutamylcysteine synthetase gene expression.

We previously reported that the activity of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis, is under both hormonal and cell density regulation in cultured rat hepatocytes. Specifically, the addition of insulin or hydrocortisone to culture media or the lowering of the initial plating cell density increased cell GSH by increasing the activity of GCS. In the present study, we examined the molecular mechanism of these effects. To determine whether the increase in GCS activity is associated with an increase in GCS heavy subunit (GCS-HS) mRNA expression, the steady state mRNA levels of GCS-HS were examined with the use of Northern blots. After 24-hr treatment of high density (0.6 x 10(5) cells/cm2) cultured rat hepatocytes with insulin (1 micrograms/ml) or hydrocortisone (50 nM), the steady state GCS-HS mRNA level increased by approximately 1-2 fold. When the plating density was decreased to 0.1 x 10(5) cells/cm2, the steady state GCS-HS mRNA level also increased by 1-2 fold 24 hr later. An increase in the steady state GCS-HS mRNA level was found within 4 hr of either hormonal treatment or cell density manipulation. The increase in steady state GCS-HS mRNA level resulted from increased gene transcription, as the transcriptional rates of GCS-HS after hormonal or cell density manipulation were increased by 2-3-fold, whereas the rates of GCS-HS mRNA degradation remained unchanged. Western blotting confirmed the increase in GCS-HS protein level after hormone treatment or lowering of plating cell density. When examined in vivo, the steady state GCS-HS mRNA level decreased by 50% in a rat in which diabetes had been induced with streptozotocin for 1 week; this was prevented with insulin replacement. In summary, GCS-HS gene expression is under both hormonal and cell density regulation.

Amino Acid Sequence↗

PubMed1976

Streptococcal infections in sucking pigs. 1. Epidemiological investigations.

The investigation comprises 5,811 litters and 55,641 liveborn pigs. Among the pigs which died before weaning, infection with haemolytic streptococci (HS) was determined in 1,338. This corresponds to 2.4 per cent of the liveborn pigs (Table I). In the examined herds the incidence of fatal cases with HS infections ranged from 1.2 to 3.8 per cent. The highest incidence was observed in litters born by gilts (Table II) and in litters with more tha 11 pigs (Table III). HS infections were identified in 17.5 per cent of the examined litters, and in most of the litters only one pig was lost (Table IV). A marked seasonal variation was observed, with a predominance of HS during the winter period (Table V). Analyses of the influence of some possible determinative factors revealed the highest incidence among the progeny of sows which were housed separately during pregnancy (Table VI), whereas herd size and the hygiene level did not exert any influence on the frequency of HS. The incidence of fatal HS infections was higher in herds where tail docking and clipping of the canine teeth were performed than in herds where these procedures were omitted (Table VII). Most of the death with HS infections (53.8 per cent) occurred among pigs less than 14 days of age (Table VIII). The main diagnoses of the pigs with streptococcal infections are presented in Table IX. A survey of the isolation frequency of HS from various organs is given in Table X. The most frequently isolated serogroups were group-C (40.2 per cent) and group-L (31.3 per cent) (Table XI). Preliminary investigations on the source of infection and the pathogenesis are reported (Tables XII and XIII). In two herds, 8 of 10 sows carried HS on mucosal surfaces at the time of farrowing, and in 6 of 10 examined litters, HS bacteraemias were observed in pigs during the first days of life. Furthermore, an analysis showed that part of the sowa (109) lost pigs with HS infections in several (2-6) subsequent litters (Tables XIV) and XV).

Animal Husbandry↗

PubMed2005

Alcohol- vs hypertonic saline-assisted laser-assisted subepithelial keratectomy.

OBJECTIVE: To evaluate the predictability and safety of hypertonic saline (5% sodium chloride) [correction]-assisted laser-assisted subepithelial keratectomy (HS-LASEK) vs 20% alcohol-assisted LASEK (A-LASEK). SETTING: American Laser Medical Center, Rishon Le-Zion, Israel. METHODS: Fifty-two consecutive eyes (26 patients) were randomized to HS-LASEK (30 eyes) and A-LASEK (22 eyes) groups. The patients' eyes were examined, refracted, and photographed at 1 day, 5 days, 2 weeks, and 1 month postoperatively by a masked physician. Corneal topography and confocal examination were performed before and 1 month after surgery. MAIN OUTCOME MEASURES: The predictability, accuracy, and visual recovery of HS-LASEK vs A-LASEK in the first postoperative month. RESULTS: The accuracy of HS-LASEK was similar and sometimes better than A-LASEK. Two weeks after surgery, 17 eyes (57%) in the HS-LASEK group and 10 eyes (46%) in the A-LASEK group were within +/- 0.5 diopter of the intended refractive correction (P<.05). At all time points eyes in the HS-LASEK group had better best-corrected visual acuity than eyes in the A-LASEK group, although the uncorrected visual acuity was similar. Eighteen (61%) of the HS-LASEK-treated eyes and 12 (55%) of the A-LASEK-treated eyes had an epithelial defect after surgery. The HS-LASEK-treated eyes had significantly larger epithelial defects. The resolution of the defects was faster in the HS-LASEK-treated eyes (mean +/- SD, 4.5 +/- 0.4 days vs 5.8 +/- 0.2 days, P = .002). The subepithelial scar was thicker in A-LASEK-treated eyes. CONCLUSIONS: Hypertonic saline-assisted LASEK provides good postoperative accuracy, safety, and a similar rate of complications. In view of recent evidence regarding the epithelial toxic effects of alcohol, HS-LASEK might be a better treatment alternative.

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