The role of XRCC6/Ku70 in nasopharyngeal carcinoma.

The association between XRCC6/Ku70, an upstream player in the DNA double-strand break repair system, and the risk of nasopharyngeal carcinoma (NPC) was examined. In this case-control study, 176 NPC patients and 352 cancer-free controls were genotyped, and the associations of XRCC6 promoter T-991C (rs5751129), promoter G-57C (rs2267437), promoter G-31A (rs132770), and intron 3 (rs132774) polymorphisms with NPC risk were evaluated. NPC tissue samples were also assessed for their XRCC6 mRNA and protein expression by real-time quantitative reverse transcription PCR and Western blotting, respectively. With regard to the XRCC6 promoter T-991C, the TC and CC genotypes were associated with a significantly increased risk of NPC compared with wild-type TT genotype (adjusted odds ratio 2.02 and 3.42, 95% confidence interval 1.21-3.32 and 1.28-8.94, P=0.0072 and 0.0165, respectively). The mRNA and protein expression levels for NPC tissues revealed significantly lower XRCC6 mRNA and protein expression in the NPC samples with TC/CC genotypes compared to those with the TT genotype (P=0.0210 and 0.0164, respectively). These findings suggest that XRCC6 may play an important role in the carcinogenesis of NPC and could serve as a chemotherapeutic target for personalized medicine and therapy.

Bilateral thermal capsulotomy with MR-guided focused ultrasound for patients with treatment-refractory obsessive-compulsive disorder: a proof-of-concept study.

Despite optimal pharmacotherapy and cognitive-behavioral treatments, a proportion of patients with obsessive-compulsive disorder (OCD) remain refractory to treatment. Neurosurgical ablative or nondestructive stimulation procedures to treat these refractory patients have been investigated. However, despite the potential benefits of these surgical procedures, patients show significant surgery-related complications. This preliminary study investigated the use of bilateral thermal capsulotomy for patients with treatment-refractory OCD using magnetic resonance-guided focused ultrasound (MRgFUS) as a novel, minimally invasive, non-cranium-opening surgical technique. Between February and May 2013, four patients with medically refractory OCD were treated with MRgFUS to ablate the anterior limb of the internal capsule. Patients underwent comprehensive neuropsychological evaluations and imaging at baseline, 1 week, 1 month and 6 months following treatment. Outcomes were measured with the Yale-Brown Obsessive-Compulsive Scale (Y-BOCS), the Hamilton Rating Scale for Depression (HAM-D) and the Hamilton Rating Scale for Anxiety (HAM-A), and treatment-related adverse events were evaluated. The results showed gradual improvements in Y-BOCS scores (a mean improvement of 33%) over the 6-month follow-up period, and all patients showed almost immediate and sustained improvements in depression (a mean reduction of 61.1%) and anxiety (a mean reduction of 69.4%). No patients demonstrated any side effects (physical or neuropsychological) in relation to the procedure. In addition, there were no significant differences found in the comprehensive neuropsychological test scores between the baseline and 6-month time points. This study demonstrates that bilateral thermal capsulotomy with MRgFUS can be used without inducing side effects to treat patients with medically refractory OCD. If larger trials validate the safety, effectiveness and long-term durability of this new approach, this procedure could considerably change the clinical management of treatment-refractory OCD.

Peripheral compressing artifacts in brain tissue from stereotactic biopsy with sidecutting biopsy needle: a pitfall for adequate glioma grading.

AIMS: The stereotactic brain biopsy is an essential diagnostic procedure in modern neurologic patient management. A side-cutting biopsy needle is one of the most widely used needle types. Recently we found a characteristic tissue artifact named "peripheral compressing artifact" in the brain tissues biopsied using a side-cutting needle of Leksell's system. We investigate prevalence, possible cause and its clinical implication of this type of artifact. MATERIALS AND METHODS: We examined the biopsies from 80 patients (44 cases of gliomas, 13 lymphomas, 7 germ cell tumors, 2 other tumors, 1 metastatic carcinoma, 4 non-tumorous conditions such as demyelinating disease and 8 non-diagnostic) in the stereotactic biopsy group with a suspected brain tumor, who underwent a stereotactic brain biopsy using side-cutting needle of Leksell's system. We also evaluated 16 cases of open brain biopsies without Leksell's system as a control group. RESULTS: The artifact is a semi-circular or band-like tissue compression in the periphery of the biopsied tissue. This artifact was found in 30 (37.5%) out of 80 cases and 57 (11.9%) out of 477 biopsied pieces. It might be produced during rotating of the inner cannula of the biopsy needle. Histologically, it might be misinterpreted as "hypercellular", "spindle", "well circumscribed", or rarely as "pseudopalisading" especially in glioma. CONCLUSIONS: Awareness of this artifact would help making the appropriate pathological diagnosis for glioma.

An evaluation of the contributions by fresh gas flow rate, carbon dioxide concentration and desflurane partial pressure to carbon monoxide concentration during low fresh gas flows to a circle anaesthetic breathing system.

BACKGROUND AND OBJECTIVE: Numerous in vitro studies have shown that volatile anaesthetics react with desiccated carbon dioxide (CO2) absorbents to produce carbon monoxide (CO). The effects of anaesthetic concentration, fresh gas flow rate, and the hydration of absorbent or the excretion of CO2 by patients on CO production have also been investigated. This work aims to identify the most significant one of these factors on CO concentration in a low-flow anaesthesia system, without control of the hydration of the absorbents. METHODS: A simulated clinical circle anaesthetic breathing system was used to study the CO concentration under various conditions. Desflurane was used at three different concentrations. Two CO2 flow rates and three fresh gas flow rates were used. The absorbent temperatures and hydration were measured simultaneously. RESULTS: Desflurane degraded to produce CO in the breathing tube, when the CO2 absorbents were not dried beforehand. In this imitation clinical low-flow setting, fresh gas flow affected the CO production more than the CO2 did (31.7% vs. 9.5%). The actual desflurane partial pressure was not a significant factor. The CO2 flow rate explained 18.2% and 54.0% of the variation of the absorbent hydration changes (%) and temperature, respectively. CONCLUSIONS: In clinical practice, the CO2 production varies among patients and is uncontrollable, but markedly affects CO production. The only controllable factor is the fresh gas flow rate if the ultimate goal is to reduce the undesirable exposure of patients to CO from the breathing tube according to this bench model without counting the oxygen consumption.

Elective radiation therapy for high-risk malignant melanomas.

PURPOSE: Local-regional recurrence rates of 30%-50% have been reported after resection of high-risk malignant melanomas (multiple node involvement, extracapsular spread, deep invasion, recurrent disease, and/or microscopically involved margins). Recently, we have been offering elective radiation therapy, after definitive surgery, to selected patients who have high-risk malignant melanomas. We herein report our initial results. PATIENTS AND METHODS: From 1993 to 1999, 40 patients who underwent surgery for high-risk malignant melanomas (multiple involved lymph nodes [21 patients]; close or microscopically involved surgical margins [nine patients]; extracapsular extension [six patients]; previously resected, recurrent disease [three patients]; and/or primary tumors more than 4 mm thick [four patients]) received elective radiation therapy. Thirty-six patients received 3000 cGy in five fractions (600 cGy per fraction given twice weekly), and four patients received 3600 cGy in six fractions. RESULTS: At a median follow-up of 18.4 months (range, 3.8-74.1 months), the actuarial 5-year local-regional control rate was 84%. Systemic recurrence rates in these patients were similar to those reported for this subset of patients, and the actuarial overall survival rate at 5 years was 39%. Acute toxicity was limited to erythema of the skin and, in one instance, probable cellulitis, with no late sequelae. DISCUSSION: Elective radiation therapy (600 cGy per fraction for five or six fractions) effectively controlled residual subclinical disease after surgery; however, better adjuvant systemic therapies need to be designed to eliminate distant metastases and to alter survival rates.

Laparoscopic subsegmentectomy for hepatocellular carcinoma with cirrhosis.

BACKGROUND/AIMS: Laparoscopic liver resection is feasible for both benign and malignant disease with today's laparoscopic techniques and technology. Location of the tumor at the edge of segment 3, 4, 5, or 6 of our patients makes them an ideal candidate for laparoscopic resection. METHODOLOGY: There were 9 patients who underwent laparoscopic subsegmentectomy for hepatocellular carcinoma with cirrhosis. They were classified as Child A in 6 and B in 3 patients. Hepatitis B was found in 5 and Hepatitis C in 4 cases. Preoperative diagnosis of hepatocellular carcinoma was completed in 7 and definitive histologic diagnosis from frozen section in 2 cases. All 9 patients underwent subsegmentectomy and removal of the tumor with non-tumor cirrhotic liver with a distance of 10 mm at the least margin. Laparoscopic ultrasound allows exact localization of lesions and achievement of adequate resection margin. RESULTS: Those patients resumed a full diet on the 2nd-3rd day after the operation and were discharged home on day 4-7 with no complications but one had prolonging discharge due to ascitis from a drainage tube. Finally, the ascitis was controlled by medications for 1 week. All patients had high postoperative satisfaction. CONCLUSIONS: Laparoscopic liver resection is a procedure of significant risk and technically demanding. Therefore, it should be performed only by experienced liver surgeons with a high level of laparoscopic skill and in the carefully selected patient.

Tissue-specific cancer-related serpin gene cluster at human chromosome band 3q26.

Approximately one quarter of the identified human serpin genes are cancer-related and clustered mainly at two distinct loci: 6p25 and 18q21. We have studied a novel serpin gene cluster at 3q26 containing at least two recently identified members: the pancreas-specific protease inhibitor, pancpin (PI14), and the brain-associated protease inhibitor, neuroserpin (PI12). In this, unlike a previous study, both PI14 and PI12 at 3q26 were found to consist of 9 exons and 8 introns and to share a perfectly conserved gene organization whose pattern is very different from that of the ov-serpin family. This distinct pattern appears identical in the genomic structures of human plasminogen activator inhibitor-1 (PAI1) at 7q21 and protease nexin 1 (PI7) at 2q33-35, confirming that these four genes in three different chromosomes form a discrete subset within the serpin superfamily. As in the other three members whose gene expression is altered during tumorigenesis, PI12 expression was found to be down-regulated in tumor brain tissues and in two brain cancer cell lines: U-87 MG and H4. By screening genomic libraries, we isolated two overlapping clones showing that the marker SGC32223 (centromere) is located within intron F of PI12 and the marker WI-10077 (telomere) is located downstream of the 3'-flanking region of PI14. This finding indicates that the distance between human PI14 and PI12 is approximately 100 kb, and hence we speculate that other tissue-specific cancer-related serpin genes are likely to reside within this 3q26.1 cluster region.

Implications for function and therapy of a 2.9 A structure of binary-complexed antithrombin.

The crystal structure of a binary complex of human antithrombin with a peptide of the same sequence as its reactive loop (P14-P3) has been determined at 2.9 A. The peptide binds as the middle strand s4A in the A beta-sheet, homologously to that of the reactive loop in the latent and cleaved forms of antithrombin. Peptide binding results in the complete expulsion of the hinge region of the loop from the A beta-sheet although the conformation differs from that of heparin-activated antithrombin. The 36-fold increase in the rate of reaction of the binary complex with factor Xa indicates that full loop expulsion alone is not sufficient for complete heparin activation of antithrombin but that this is also dependent on the overall conformation of the molecule. Previous studies have demonstrated that reactive loop peptides can block or reverse the polymerisation of serpins associated with cirrhosis and thrombosis. The antithrombin binary complex structure defines the precise localisation of the blocking peptide in a serpin and provides the basis for rational drug design for mimetics that will prevent polymerisation in vivo and so ameliorate the associated disease.

Mechanisms of antithrombin polymerisation and heparin activation probed by the insertion of synthetic reactive loop peptides.

Incubation of antithrombin with a series of synthetic reactive loop peptides showed that 6-mer and 7-mer peptides, P14-P9 and P14-P8 of antithrombin respectively, induced loop-sheet polymerisation and binary complex formation. These peptides are likely to anneal to the upper part of the dominant A-sheet, favouring sheet opening and allowing insertion of a second reactive loop in the lower part of the A-sheet to form polymers. The insertion of longer peptides filled the A-sheet beyond the P7 position and prevented polymerisation. Heparinised antithrombin was more resistant to polymerisation and peptide insertion, indicating that heparin induces a conformational change that closes the A-sheet and expels the reactive loop.

The effects of reactive centre loop length upon serpin polymerisation.

The clinical effects of serpin polymerisation include thromboembolism, emphysema, and liver disease. A through understanding of serpin polymerisation mechanisms and the structures involved will permit the rational design of therapeutic polymerisation inhibitors. Here we show that serpin polymerisation can be delayed by extending the length of the serpin reactive centre loop. The heat stability of three chimeric serpins was examined. One of them, an active alpha 1-antitrypsin variant with a reactive centre loop C-terminal extension of four amino acid residues, was shown to have increased resistance to inactivation by polymerisation. This variant could also form serpin/peptide binary complexes with a reactive centre loop peptide, which indicates that the increase in thermostability was not due to the A-beta-sheet being unable to accept reactive centre loop residues, an essential requirement for polymerisation. Rather, we conclude that the additional residues within the reactive centre loop delay the release of strand 1C from the C-sheet, a process essential for polymer formation.

Structure-activity relationship of coumarins in xanthine oxidase inhibition.

Esculetin(4), umbelliferone(7-hydroxycoumarin)(3) and 7-hydroxy-4-methyl coumarin(8) are strong xanthine oxidase inhibitors (IC50 = 20.91, 43.65 and 96.70 microM respectively). Based on this observation, the structure of 7-hydroxy coumarin(3) plays a very important role in xanthine oxidase (XO) inhibition. The 6-hydroxy group present in the molecule of 7-hydroxy coumarin, e.g. esculetin(4) enhanced the activity, whereas substitution by the 6-methoxy group, e.g. scopoletin (5), reduced the inhibitory effect. Furthermore, 6-glycoside group present in the molecule of 7-hydroxy coumarin, e.g. esculin (6,7-dihydroxy coumarin 6-glucoside)(12) strongly decreased the inhibitory effect as well as scoparone(6), the fully methylated derivative of esculetin (4). In contrast to 7-hydroxy coumarin(3), however, 4-hydroxy coumarin(13) showed only a weak effect on XO inhibition. 4-Substituent present in the molecule of 7-hydroxycoumarin also reduced the activity but the degree of reduction depended on the substituents: 7-hydroxy-4-methylcoumarin (8) < 7-hydroxycoumarin-4-acetic acid (7) < 7-hydroxy-4-trifluoromethylcoumarin (9). Their percent inhibition at 100 microM was 62.47, 38.46 and 26.84% respectively. 8-substituent present in the molecule of 7-hydroxy coumarin (3), such as 7,8-dihydroxy-6-methoxycoumarin(10) and fraxin(7-hydroxy-6-methoxycoumarin 8-glucoside)(11) reduced the activity as compared with scopoletin (5). Their percent inhibition at 100 microM was 18.4 and 6.9% respectively, which indicated that the more bulky the 8-substituted in the structure, the weaker the inhibitory activity on XO. 3,4,8-Trimethyl-7-hydroxycoumarin(14) which substitution by the methyl at 3,4 & 8 in the structure of 7-hydroxycoumarin(3) also reduced the activity as compared with 7-hydroxycoumarin(3). It seems that the double bond in the structure of coumarin(1) played an important role in the activity as compared with coumarin(dihydrocoumarin)(2). The apparent inhibition constants(Ki) of esculetin(4), umbelliferone (3) and 7-hydroxy-4-methylcoumarin(8) were 2.056, 21.683 and 4.86 microM respectively and induced competitive, uncompetitive and a mixed type of inhibition of the enzyme with respect to the substrate xanthine.

Inhibitory effects of phenolic carboxylic acid analogues on xanthine oxidase.

Nineteen phenolic carboxylic acid analogues were tested for the effects on xanthine oxidase inhibition. 2,2',4,'4'-Tetrahydroxybenzophenone and 2,3,4-trihydroxybenzoic acid displayed the strongest activities (IC50 = 38.70 microM, IC50 = 90.16 microM respectively). Their apparent inhibition constants (Ki) were 7.052 and 0.535 microM respectively, and induced mixed type and competitive type inhibitions respectively with respect to the substrate xanthine.

Coexisting sharp ductal angulation with intrahepatic biliary strictures in right hepatolithiasis.

OBJECTIVE: To investigate the clinical characteristics of a coexisting sharp ductal angulation (< 90 degrees) with biliary stricture and to evaluate the difficulties it imposes in the management of retained or recurrent hepatolithiasis. DESIGN: Case-controlled study. SETTING: A referral center. PATIENTS: Eighteen consecutive patients having right-sided hepatolithiasis and a coexisting sharp ductal angulation associated with biliary stricture (group 1) were compared with 84 patients matched with sex, age, and conditions of hepatolithiasis and intrahepatic biliary stricture(s) but no sharp angulated duct (group 2). INTERVENTION: Postoperative cholangioscopic management (electrohydraulic lithotripsy or other lithotripsy, lithotomy, balloon dilation, biopsy, etc, via T-tube tract or percutaneous transhepatic route). MAIN OUTCOME MEASURES: Sessions of manipulations, incidence of complications associated with interventions or disease, and mortality were compared. RESULTS: Patients of group 1 needed more sessions of postoperative manipulation of stones and strictures (13.7 +/- 4.2 vs 8.0 +/- 2.3; P < .001). During management, there was a significantly increased vulnerability of severe and/or recurrent cholangitis (66.7% vs 9.5%; P < .001), septic shock (77.8% vs 11.9%; P < .001), liver abscess (55.6% vs 7.1%; P < .001), or massive hemobilia (33.3% vs 7.4%) in group 1 than in group 2. Their risks of coexisting secondary biliary cirrhosis (55.6% vs 9.5%; P < .001) and/or cholangiocarcinoma (16.6% vs 2.4%; P < .04) and mortality (27.8% vs 4.8%; P < .01) were also significantly higher in group 1. CONCLUSION: Our results suggest that the coexisting sharp ductal angulation with biliary strictures in right-sided hepatolithiasis is a distinct difficult clinical entity in the field of biliary tract calculi.

Inhibitory effects of phenolics on xanthine oxidase.

The stems of Bougainvillea spectabillis Wild (Nyctaginaceae) have been used in folk medicine against hepatitis. Spinasterol, 22, 23-dihydrospinasterol and caffeic acid were isolated from the plant stems and characterized. Caffeic acid has not been previously isolated from this plant but spinasterol has been isolated from the leaves. Caffeic acid was found to be the active principle exhibiting strong inhibition of xanthine oxidase in this study (IC50 = 39.21 microM). In order to study the structure-activity relationship of the phenolics as regards xanthine oxidase inhibition, twelve naturally occurring phenolics (esculetin, scopoletin, scoparone, barbaloin, berberine chloride, sinomenine, osthole, paeonol, honokiol, magnolol, methyleugenol and 6-gingerol) were tested for their inhibitory effects on xanthine oxidase. The results showed that esculetin displayed the strongest activity (IC50 = 28.4 microM), and induced competitive inhibition of the enzyme with respect to the substrate xanthine. The apparent inhibition constant (Ki) of esculetin was 2.369 x 10(-6) M. Since xanthine oxidase serum levels are increased in hepatic and brain tumors, caffeic acid and esculetin should be tested as anti-hepatitis or/and anticancer agents.

Inhibitory effects of flavonoids on xanthine oxidase.

The stems of Bougainvillea spectabillis Wild (Nyctaginaceae) have been used in folk medicine for hepatis, and spinasterol and quercetin were isolated and characterized from the plant leaves in this study. These constituents have not been previously isolated from Bougainvillea spectabillis W. Quercetin, the flavonoid, was found as active principle because it showed a strong activity on xanthine oxidase inhibition (IC50 = 7.23 microM) in this study as well as in the literature. Since xanthine oxidase serum levels are increased in hepatitis and tumoral brain tissues, quercetin may be used for remission of hepatitis or brain tumor. In order to study the structure-activity relationship of the flavonoids as regards xanthine oxidase inhibition, nine naturally occurring flavonoids have been tested the inhibitory effects on xanthine oxidase, such as baicalein, baicalin, capillarisin, d-catechin, d-epicatechin, hesperidin, liquiritin, puerarin and wogonin. The results showed that baicalein displayed the strongest activity (IC50 = 9.44 microM), followed by wogonin (IC50 = 52.46 microM) and then baicalin (IC50 = 71.73 microns). Baicalein induced uncompetitive inhibition of the enzyme with respect to xanhtine and the apparent inhibition constant (Ki) was 2.48 x 10(-6) M.

Expression of epithelial alkaline phosphatase in segmentally iterated bands during grasshopper limb morphogenesis.

Although the study of rostral-caudal segmentation of the insect body has been a rich source of information about embryonic pattern formation, relatively little is known of the process of proximal-distal segmentation of insect appendages. Here we demonstrate that during the period of limb segmentation, five segmentally iterated, sharply demarcated bands of cell surface alkaline phosphatase activity are expressed in embryonic grasshopper limbs. These bands span each intersegmental boundary in the limb as well as one boundary within the tarsus. Within appendages, expression is restricted to epithelial cells, where activity is present on both apical and basolateral surfaces. This epithelial alkaline phosphatase remains active at neutral pH, is insensitive to levamisole inhibition, and is strongly inhibited by nucleoside monophosphates. Treatment of embryos with phosphatidylinositol-specific phospholipase C releases almost all visible chromogenic activity, indicating that the epithelial alkaline phosphatase is anchored to the plasma membrane by glycosyl-phosphatidylinositol. When material released by phosphatidylinositol-specific phospholipase C is separated on native polyacrylamide gels, a single broad band of enzymatic activity is detected following incubation with substrate. A polyclonal antiserum raised against a 55 x 10(3) M(r) alkaline phosphatase from shrimp recognizes a single band of 56 x 10(3) M(r) on immunoblots of grasshopper membrane proteins. The spatially restricted expression of epithelial alkaline phosphatase suggests that it may be involved in epithelial cell rearrangements or shape changes associated with limb segmentation and morphogenesis. It also may contribute to definition of axon routes in the limb, since pioneer afferent growth cones turn at, and migrate along, the edge of one alkaline phosphatase-expressing epithelial domain.

Disruption of pioneer growth cone guidance in vivo by removal of glycosyl-phosphatidylinositol-anchored cell surface proteins.

Cell surface proteins anchored to membranes via covalently attached glycosyl-phosphatidylinositol (GPI) have been implicated in neuronal adhesion, promotion of neurite outgrowth and directed cell migration. Treatment of grasshopper embryos with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that cleaves the GPI anchor, often induced disruptions in the highly stereotyped migrations of peripheral pioneer growth cones and afferent neuron cell bodies. In distal limb regions of embryos treated with PI-PLC at early stages of pioneer axon outgrowth, growth cones lost their proximal orientation toward the central nervous system (CNS) and turned distally. Pioneer growth cones in treated limbs also failed to make a characteristic ventral turn along the trochanter-coxa (Tr-Cx) segment boundary, and instead continued to grow proximally across the boundary. Treatment at an earlier stage of development caused pre-axonogenesis Cx1 neurons to abandon their normal circumferential migration and reorient toward the CNS. None of these abnormal phenotypes were observed in limbs of untreated embryos or embryos exposed to other phospholipases that do not release GPI-anchored proteins. Incubation of embryos with PI-PLC effectively removed immunoreactivity for fasciclin I, a GPI-anchored protein expressed on a subset of neuronal surfaces. These results suggest that cell surface GPI-anchored proteins are involved in pioneer growth cone guidance and in pre-axonogenesis migration of neurons in the grasshopper limb bud in vivo.

Immunization strategies for the production of rat monoclonal anti-idiotope antibodies.

Anti-idiotypic antibodies are powerful reagents for the study of immunoregulation, and have potential interest as vaccines against tumors and infectious diseases. Three immunization strategies for the production of rat monoclonal anti-idiotope antibodies have been compared in this paper. Male Wistar rats were immunized i.p. and at multiple subcutaneous sites with 750 micrograms of purified monoclonal antibody against Plasmodium falciparum for three times and subsequently boosted by (1) intraperitoneal injection with 750 micrograms of the immunogen, (2) intravenous inoculation with 400 micrograms of the IgG, and (3) intrasplenic immunization with 200 micrograms of the idiotype. With the intraperitoneal boost method, the frequency of hybrids with anti-idiotope activity was 0.3-0.9% with 62.8-85.2% of the seeded wells containing hybrids. In the intravenous boost group, the percentage of hybrids demonstrating anti-idiotope activity increased to 11.0-13.3% with 80.2-97.9% of the hybrid efficiency. When immunized by the intrasplenic boost route, the frequency of anti-idiotope hybrids generated rose to 12.9-16.4% with 82.3-96.6% of the hybrid efficiency. There was no obvious effect of the boost immunizing methods on the generation of rat monoclonal anti-mouse IgG antibodies. These results indicated that the multiple-site immunization followed by intravenous or intrasplenic boost injection was an appropriate immunizing method for the production of monoclonal anti-idiotope antibodies.

Cholesterol requirement for growth of IR983F and P3X63-Ag8-U1 myeloma cells in serum-free medium.

Cholesterol, a major lipid component of the plasma membrane, is thought to have profound effects on the structure and function of cells. Most animal tissues are capable of synthesizing cholesterol de novo from acetate; however, there are relatively few mammalian cells in vitro expressing an absolute requirement for an exogenous source of cholesterol. In this paper, it was shown that both IR983F (983) rat myeloma cells and P3X63-Ag8-U1 (P3U1) mouse myeloma cells which had been cultivated in serum-free medium containing cholesterol for more than 6 months still required cholesterol in vitro for growth in serum-free medium. Optimal growth of 983 and P3U1 occurred in cholesterol concentrations of 15 and 5 micrograms/ml, respectively. Moreover, it was demonstrated that the cholesterol could be replaced by human low density lipoprotein in a concentration of 10 micrograms/ml but not by mevalonic acid lactone. In contrast to the parental myeloma cells, hybridoma cells derived from the mouse myeloma cells which had been cultivated in serum-free medium containing cholesterol for more than 6 months did not require cholesterol.