The plasma membrane-associated protein RS1 decreases transcription of the transporter SGLT1 in confluent LLC-PK1 cells.

Previously we cloned RS1, a 67-kDa polypeptide that is associated with the intracellular side of the plasma membrane. Upon co-expression in Xenopus laevis oocytes, human RS1 decreased the concentration of the Na(+)-D-glucose co-transporter hSGLT1 in the plasma membrane (Valentin, M., Kühlkamp, T., Wagner, K., Krohne, G., Arndt, P., Baumgarten, K., Weber, W.-M., Segal, A., Veyhl, M., and Koepsell, H. (2000) Biochim. Biophys. Acta 1468, 367-380). Here, the porcine renal epithelial cell line LLC-PK1 was used to investigate whether porcine RS1 (pRS1) plays a role in transcriptional up-regulation of SGLT1 after confluence and in down-regulation of SGLT1 by high extracellular D-glucose concentrations. Western blots indicated a dramatic decrease of endogenous pRS1 protein at the plasma membrane after confluence but no significant effect of D-glucose. In confluent LLC-PK1 cells overexpressing pRS1, SGLT1 mRNA, protein, and methyl-alpha-D-glucopyranoside uptakes were drastically decreased; however, the reduction of methyl-alpha-D-glucopyranoside uptake after cultivation with 25 mm D-glucose remained. In confluent pRS1 antisense cells, the expression of SGLT1 mRNA and protein was strongly increased, whereas the reduction of SGLT1 expression during cultivation with high D-glucose was not influenced. Nuclear run-on assays showed that the transcription of SGLT1 was 10-fold increased in the pRS1 antisense cells. The data suggest that RS1 participates in transcriptional up-regulation of SGLT1 after confluence but not in down-regulation by D-glucose.

Atraumatic osteonecrosis of the patella.

Atraumatic secondary osteonecrosis of the patella is a rare entity and has been reported in only several case reports. The purpose of this study was to define the clinical and radiographic characteristics and outcome of this disease. Osteonecrosis of the patella was found in 25 knees in 19 patients. The mean age was 42 years (range, 21-63 years). Eighteen (95%) patients had greater than 2 g of lifetime corticosteroid exposure, and six (32%) patients had systemic lupus erythematosus. Osteonecrosis was found in the superior pole of the patella in all 23 knees (17 patients) that had magnetic resonance imaging. Osteonecrosis was apparent on plain radiographs in five knees (20%) in four patients, and only one knee (4%) in one patient had collapse of the posterior articulating surface of the patella. One (4%) patient presented with anterior knee pain localized to the patella. At a mean followup of 4 years (range, 2-18 years), none of the patients had an intervention that focused on treating the patellar lesion. Patellar osteonecrosis is characterized by patients with coincident lesions of the distal femur and the proximal tibia and lesions localized to the superior pole of the patella. It is a nonprogressive disease that does not warrant surgical exploration.

The transport modifier RS1 is localized at the inner side of the plasma membrane and changes membrane capacitance.

Previously we cloned membrane associated (M(r) 62000-67000) polypeptides from pig (pRS1), rabbit (rbRS1) and man (hRS1) which modified transport activities that were expressed in Xenopus laevis oocytes by the Na(+)-D-glucose cotransporter SGLT1 and/or the organic cation transporter OCT2. These effects were dependent on the species of RS1 and on the target transporters. hRS1 and rbRS1 were shown to be intronless single copy genes which are expressed in various tissues and cell types. Earlier immunohistochemical data with a monoclonal IgM antibody suggested an extracellular membrane association of RS1. In the present paper antibodies against recombinant pRS1 were raised and the distribution and membrane localization of RS1 reevaluated. After subcellular fractionation of renal cortex RS1 was found associated with brush border membranes and an about 1:200 relation between RS1 and SGLT1 protein was estimated. Also after overexpression in X. laevis oocytes RS1 was associated with the plasma membrane, however, at variance to the kidney it was also observed in the cytosol. Labeling experiments with covalently binding lipid-permeable and lipid-impermeable biotin analogues showed that RS1 is localized at the inner side of the plasma membrane. Western blots with plasma membranes from Xenopus oocytes revealed that SGLT1 protein in the plasma membrane was reduced when hRS1 was coexpressed with human SGLT1 which leads to a reduction in V(max) of expressed glucose transport. Measurements of membrane capacitance and electron microscopic inspection showed that the expression of hRS1 leads to a reduction of the oocyte plasma membrane surface. The data suggest that RS1 is an intracellular regulatory protein that associates with the plasma membrane. Overexpression of RS1 may effect the incorporation and/or retrieval of transporters into the plasma membrane.

Atraumatic osteonecrosis of the knee.

BACKGROUND: The purposes of this study were to define the clinical, demographic, and radiographic patterns of atraumatic osteonecrosis of the distal part of the femur and the proximal part of the tibia at presentation and to report the outcome of treatment of this condition. METHODS: Two hundred and forty-eight knees in 136 patients who were younger than the age of fifty-five years were treated at our institution between July 1, 1974, and September 15, 1998, for atraumatic osteonecrosis of the distal part of the femur or the proximal part of the tibia, or both. Demographic and radiographic features were characterized. The results of nonoperative treatment, core decompression, arthroscopic debridement, and total knee arthroplasty were evaluated. RESULTS: There were 106 female patients and thirty male patients, and their mean age was thirty-six years (range, fifteen to fifty-four years) at the time of diagnosis. One hundred and one patients (74 percent) had involvement of other large joints, with eighteen (13 percent) presenting initially with knee symptoms. One hundred and one patients (74 percent) had a disease that affected the immune system; sixty-seven of them had systemic lupus erythematosus. One hundred and twenty-three patients (90 percent) had a history of corticosteroid use. Technetium-99m bone-scanning missed lesions in sixteen (29 percent) of fifty-six knees. Eight (20 percent) of forty-one initially symptomatic knees treated nonoperatively had a successful clinical outcome (a Knee Society score of at least 80 points and no additional surgery) at a mean of eight years. The knees that remained severely symptomatic for three months were treated with either core decompression (ninety-one knees) or total knee arthroplasty (seven knees). Seventy-two (79 percent) of the ninety-one knees treated with core decompression had a good or excellent clinical outcome at a mean of seven years. Efforts to avoid total knee arthroplasty with repeat core decompression or arthroscopic debridement led to a successful outcome in fifteen (60 percent) of twenty-five knees. Thirty-four (71 percent) of forty-eight knees treated with total knee arthroplasty had a successful clinical outcome at a mean of nine years. CONCLUSIONS: Atraumatic osteonecrosis of the knee predominantly affects women, and in our study it was associated with corticosteroid use in 90 percent of the patients. Evaluation should include standard radiographic and magnetic resonance imaging of all symptomatic joints. Prognosis was negatively related to large juxta-articular lesions. Nonoperative treatment should be reserved for asymptomatic knees only. Core decompression was successful (a Knee Society score of at least 80 points and no additional surgery) in 79 percent of the knees in which the disease was in an early stage. Total knee arthroplasty was successful in only 71 percent of the knees.

Financial impact of a home intravenous antibiotic program on a medicare managed care program.

This study quantitates cost savings achieved by a home intravenous antibiotic (HIVA) program in a Medicare managed health care program. In 1998, 66 treatment courses of HIVA therapy were administered for a total of 1542 patient-days of therapy. The calculated cost of HIVA therapy included the actual costs of drugs, supplies, nursing and therapists' salaries, and laboratory studies. Savings were calculated based on the average daily direct variable cost (DDVC) for hospital acute unit or skilled nursing facility (SNF) care associated with the patient's discharge diagnosis-related-group. The number of days on HIVA therapy was assumed to equal the number of days in the hospital acute unit or hospital-based SNF. The average cost per day of HIVA therapy was $122, whereas average DDVC of hospital acute unit care was $798, and the average DDVC of SNF care was $541. In 1 year, the HIVA program saved our health care system $646,000-$834,000, which demonstrates that HIVA programs are powerful tools to reduce costs in Medicare managed health care programs.

High extracellular K+ during hypoxic preconditioning episodes attenuates the post-ischemic contractile and ionic benefits of preconditioning.

Hypoxic preconditioning improves contractile recovery and decreases calcium loading following ischemia and reperfusion. To test whether changing the trans-sarcolemmal K+ gradient during the preconditioning period changes preconditioning's benefits, isolated rat hearts were subjected to two, 5 min hypoxic intervals in the presence of normal K+ (5mM, NmlK-PC) or high K+ (10.3 mM, HiK-PC), separated by 5 min of normoxic reflow. Preconditioning with 5 mM K+ significantly improved developed pressure (DP) after 30 min of ischemia as compared to non-preconditioned control hearts (55.9+/-4.41% v 12.4+/-2.01% of baseline, P<0.05). DP recovery was diminished with 10.3 mM K+ (25.1+/-4.20% of baseline, P<0.05). At the end of reperfusion, cell Ca2+ trended lower in hypoxic preconditioned hearts compared with control hearts (12.9+/-1.9 v 19.4+/-2.6 micromol/g dry wt, P=0.09) and was significantly lower than high K+ hearts (22.9+/-1.4 micromol/g dry wt, P<0.006). Intracellular K+ during reperfusion was significantly higher in preconditioned compared with control hearts (P<0.02) and high K+ hearts (P<0.002) (231+/-10 v 166+/-17 v 155+/-14 micromol/g dry wt, respectively). Thus, the trans-sarcolemmal K+ gradient during the preconditioning period influences preconditioning effects; decreasing the gradient attenuates preconditioning's favorable influences on contractile recovery, cellular K+ loss, and calcium loading during reperfusion.

Oxaloacetate transport into plant mitochondria

The properties of oxaloacetate (OA) transport into mitochondria from potato (Solanum tuberosum) tuber and pea (Pisum sativum) leaves were studied by measuring the uptake of 14C-labeled OA into liposomes with incorporated mitochondrial membrane proteins preloaded with various dicarboxylates or citrate. OA was found to be transported in an obligatory counterexchange with malate, 2-oxoglutarate, succinate, citrate, or aspartate. Phtalonate inhibited all of these countertransports. OA-malate countertransport was inhibited by 4, 4'-dithiocyanostilbene-2,2'-disulfonate and pyridoxal phosphate, and also by p-chloromercuribenzene sulfonate and mersalyl, indicating that a lysine and a cysteine residue of the translocator protein are involved in the transport. From these and other inhibition studies, we concluded that plant mitochondria contain an OA translocator that differs from all other known mitochondrial translocators. Major functions of this translocator are the export of reducing equivalents from the mitochondria via the malate-OA shuttle and the export of citrate via the citrate-OA shuttle. In the cytosol, citrate can then be converted either into 2-oxoglutarate for use as a carbon skeleton for nitrate assimilation or into acetyl-coenzyme A for use as a precursor for fatty acid elongation or isoprenoid biosynthesis.

Transport of the new chemotherapeutic agent beta-D-glucosylisophosphoramide mustard (D-19575) into tumor cells is mediated by the Na+-D-glucose cotransporter SAAT1.

For beta-D-glucosylisophosphoramide mustard (beta-D-Glc-IPM), a new alkylating drug in which isophosphoramide mustard is stabilized, a higher selectivity and lower myelotoxicity was observed than for the currently used cytostatic ifosfamide. Because beta-D-Glc-IPM is hydrophilic and does not diffuse passively through the lipid bilayer, we investigated whether a transporter may be involved in the cellular uptake. A variety of cloned Na+-sugar cotransporters were expressed in Xenopus oocytes, and uptake measurements were performed. By tracer uptake and electrical measurements it was found that beta-D-Glc-IPM was transported by the low-affinity Na+-D-glucose cotransporter SAAT1, which had been cloned from pig and is also expressed in humans. At membrane potentials between -50 and -150 mV, a 10-fold higher substrate affinity (Km approximately 0.25 mM) and a 10-fold lower Vmax value were estimated for beta-D-Glc-IPM transport than for the transport of D-glucose or methyl-alpha-D-glucopyranoside (AMG). Transport of beta-D-Glc-IPM and glucose by SAAT1 is apparently performed by the same mechanism because similar sodium dependence, dependence on membrane potential, electrogenicity, and phlorizin inhibition were determined for beta-D-Glc-IPM, D-glucose, and AMG. Transcription of human SAAT1 was demonstrated in various human carcinomas and tumor cell lines. In one of these, the human carcinoma cell line T84, phlorizin inhibitable uptake of beta-D-Glc-IPM was demonstrated with substrate saturation and an apparent Km of 0.4 mM. The data suggest that the Na+-D-glucose cotransporter SAAT1 transports beta-D-Glc-IPM into human tumor cells and may accumulate the drug in the cells. They provide an example for drug targeting by employing a plasma membrane transporter.

Risk of human immunodeficiency virus (HIV) transmission by anti-HIV-negative blood components in Germany and Austria.

In order to estimate the residual risk of transfusion-transmitted HIV infection we have analyzed the data from two transfusion centers in Austria (Vienna) and Germany (Göttingen) from 1985 to 1994. In Vienna, an incidence of 1:42,000 positive anti-HIV tests in repeat donors and a prevalence of 1:7000 in first-time donors were found in 1993. In Göttingen, the indicence was 1:67,000 and the prevalence 1:7900 from 1985 to 1993. Based on a mathematical model which takes (a) the window period and (b) the false-negative rate of anti-HIV tests, as well as (c) human and operational errors into consideration, we have calculated the residual risk of HIV infection. The residual risk (third generation anti-HIV test) was found to be 1:520,000 (95% confidence interval 1:1340,000-1:210,000), and 1:900,000 (95% confidence interval 1:2340,000-1:380,000) for Vienna and Göttingen, respectively, in 1993. Look-back studies from 1985 till 1994 revealed transfusion-transmitted HIV infections in three recipients (for 1.9 million donations in Vienna) and one recipient (for 160,000 donations in Göttingen) of blood components. Based on our model, as well as on prevalence and incidence rates of HIV infection, it is also possible to predict the efficacy of additional measures introduced to further decrease the risk of transfusion-transmitted HIV infection through blood components.

The prevalence of moderate and severe FXII (Hageman factor) deficiency among the normal population: evaluation of the incidence of FXII deficiency among 300 healthy blood donors.

Factor XII (FXII) deficiency has been reported to be a risk factor for the development of arterial and venous thromboembolism. However, no data are available on the prevalence of FXII deficiency within the normal population. Measuring APTT and FXII activity, seven FXII deficiencies could be detected among 300 healthy blood donors. This corresponds to an incidence of FXII deficiency of 2.3%. On the basis of these data the prevalence of severe and mild FXII deficiency in the normal population can be estimated to be 1.5-3.0%. Assessment of FXII antigen levels revealed, that all seven FXII deficient individuals had FXII antigen levels matching the activity. One presented a severe FXII deficiency (1/300, 0.3%) without detectable FXII activity and an APTT prolongation of more than 120 s. The remaining six FXII deficiencies (6/300, 2.0%) were moderate variations with FXII activities ranging from 20-45% and less prolonged APTTs. Among the 300 healthy donors 16 (5.3%) subjects with prolonged APTTs were identified. Causes for APTT-prolongation were FXII deficiency (7/16), lupus anticoagulant (6/16), mild FVIII deficiency (1/16) and hepatic disorder (1/16). In the remaining sample (1/16) the cause for the prolongation of the APTT remained unexplained. Although 8.7% (26/300) of the donors had a positive family-history of thromboembolism (TE-FHx), none of the FXII deficient subjects were among those with positive TE-FHx.

[AIDS and blood transfusion]. / AIDS und Bluttransfusion.

In May, 1985, the Red Cross Blood Donation Center of Vienna, Lower Austria, and Burgenland started routine screening for HIV 1 antibodies; by summer 1989, about 750,000 blood donors and donations had been examined. During this period, 59 donors have been proved positive for HIV 1; they have been interviewed and the origin of the infection was explored. Since there were donors within this group, who had repeatedly given blood for many years, we carried out a follow-up study in order to find out whether the recipients had been infected by these donors before May, 1985. In addition, we give the test results of about 200,000 donors screened for HIV 1 antigen and discuss the value of such tests. Recently, 80,000 donors were screened for anti-HIV 1 and 2 antibodies; there has been no donor positive for HIV 2, so far. A much higher number of donors than those marked for confirmed western blot had to be regarded as questionable positive. In such cases, details of western blot results are listed; we discuss the problem of the evaluation of such results as well as the question of informing these donors.

[Treatment of tubal pregnancy with prostaglandins: a multicenter study]. / Behandlung der Eileiterschwangerschaft mit Prostaglandinen: eine Multizenterstudie.

In this prospective multi-centre-study, the new treatment of tubal pregnancies by means of local prostaglandin (PG) F2 alpha and systemic PG E2 application was compared to the usual surgical technique of eliminating the conceptus. In 71 patients treated with PG, the method proved to be successful in 81%; 21 patients (19%) required surgical intervention later. With an initial beta-hCG level of 2500 mIE/ml, the success rate increased to 88%. The duration of hospitalisation was significantly reduced in the PG group compared to the patients treated by primary operation (3 +/- 1 versus 6 +/- 2 days). In the PG-group, only 2 of 24 hysterosalpingograms showed tubal occlusions after treatment, whereas occlusion was present in 3 of 8 patients of the surgically treated group. Four subsequent intrauterine pregnancies in the PG-group occurred compared to none in the surgical group. PG treatment of tubal pregnancy in patients with a low initial beta-hCG value (less than 2500 mIE/ml) revealed promising results with regard to reduced postoperative morbidity and future fertility.

PIP: In this prospective, multicenter study, the new treatment of tubal pregnancies by means of local prostaglandin (PG) F2alpha and systemic PGE2 application was compared to the usual surgical technique of eliminating the conceptus. In 71 patients treated with PGs, the method proved to be successful in 81%; 21 patients (195) required surgical intervention at a later date. With an initial beta-hCG level of 2500 mIE/ml, the success rate increased to 88%. The duration of hospitalization was significantly reduced in the PG group compared to the patients treated by primary operation ()3 +or- 1 vs 6+or- 2 days). In the PG group, only 2 of 24 hysterosalpingograms showed tubal occlusions after treatment, whereas occlusion was present in 3 of 8 patients of the surgically treated group. 4 subsequent intrauterine pregnancies in the PG group occurred compared to none in the surgical group. PG treatment of tubal pregnancy in patients with a low initial beta-hCG value (2500 mIE/ml) revealed promising results with regard to reduced postoperative morbidity and future fertility. (author's)

Low-dose ethanol for inhibition of preterm uterine activity.

Inhibition of preterm labor by tocolytic agents reduces the incidence of preterm birth. Betamimetic drugs are the most widely used tocolytic. However, they are from time to time contraindicated, their administration involves potential risks for mother and fetus, and their tocolytic effect is sometimes insufficient. Ethanol infused in intoxicating doses was the first clinically useful tocolytic agent, but because of its potential risks to the fetus, its use is now infrequent. We have examined the efficacy of ethanol infused at much lower rates, alone or in combination with ritodrine, to inhibit preterm uterine contractions. Fifty-four women between 20 and 36 weeks of gestation participated in the study. In 14, betamimetics were contraindicated and they were given ethanol alone. In 38, ritodrine infusions (0.2 gm/min) had failed to arrest preterm labor and they were then given ethanol with ritodrine (0.2 gm/min) The dose of ethanol was 0.11 gm/kg/hour on the average, which is approximately equivalent to the metabolic rate. Altogether, 64 treatments were given, 14 with ethanol alone and 50 combined with 0.2 gm/min ritodrine. In 81% of the treatments, contractions were suppressed within 15.3 hours on the average. Preterm birth (infant less than 2500 gm) was prevented in 70% of the patients. No adverse effects attributable to ethanol were observed. Treatment with low-dose intravenous infusion of ethanol is a cheap, efficacious, and low-risk method to stop preterm uterine activity and is clinically useful for prevention of preterm birth. Possible mechanism of this effect is discussed.