Haplotyping the human T-cell receptor beta-chain gene complex by use of restriction fragment length polymorphisms.

We have studied the genetic segregation of human T-cell receptor beta-chain (TCR beta) genes on chromosome 7q in 40 CEPH (Centre d'Etude du Polymorphisme Humain) families by using restriction fragment length polymorphisms (RFLPs). We constructed haplotypes from eight RFLPs by using variable- and constant-region cDNA probes, which detect polymorphisms that span more than 600 kilobases of the TCR beta gene complex. Analysis of allele distributions between TCR beta genes revealed significant linkage disequilibrium between only 6 of the 28 different pairs of RFLPs. This linkage disequillibrium strongly influences the most efficient order to proceed for typing of these RFLPs in order to achieve maximum genetic informativeness, which in this study revealed a 97.3% level of heterozygosity within the TCR beta gene complex. Our results should provide new insight into recent reports of disease associations with the TCR beta gene complex and should assist in designing future experiments to detect or confirm the existence of disease-susceptibility loci in this region of the human genome.     

Analysis of refractory period after exercise and eucapnic voluntary hyperventilation challenge

We compared specific airway conductance (SGaw) and the FEV1 after repetitive exercise or repetitive eucapnic voluntary hyperventilation (EVH) challenges. Replicate challenges were matched in terms of inspired air conditions and minute ventilations (VE) in order to determine the degree of refractoriness after each type of challenge in patients with exercise-induced asthma. Ten patients exercised or hyperventilated dry, room temperature air at matched VE on two study days. When the patients FEV1 had returned to 90% of baseline or better, or at 3.75 h if FEV, had not returned to 90% of baseline, patients repeated the identical exercise or the EVH challenge. Minimum FEV1 values expressed as a percent of predicted FEV1 after the first and second exercise challenges were 52 +/- 16 and 58 +/- 17, respectively, which were statistically different (p less than 0.001; paired t test). Minimum FEV1 values after the first and second EVH challenges were 52 +/- 13 and 59 +/- 9% of predicted, respectively, which were also statistically different (p less than 0.01; paired t test). Seven of 10 subjects demonstrated higher SGaw values after the second exercise challenge compared with the first challenge, whereas eight of 10 subjects showed higher SGaw values after the second EVH challenge compared with the first challenge. Paired t test analysis indicated that percent protection, measured by FEV1, was similar after either type of challenge. We conclude that replicate exercise or EVH challenges with similarly matched inspired air conditions and VE induce similar degrees of refractoriness.(ABSTRACT TRUNCATED AT 250 WORDS)     

A role for autophagy in long‐term spatial memory formation in male rodents

A hallmark of long‐term memory formation is the requirement for protein synthesis. Administration of protein synthesis inhibitors impairs long‐term memory formation without influencing short‐term memory. Rapamycin is a specific inhibitor of target of rapamycin complex 1 (TORC1) that has been shown to block protein synthesis and impair long‐term memory. In addition to regulating protein synthesis, TORC1 also phosphorylates Unc‐51‐like autophagy activating kinase‐1 (Ulk‐1) to suppress autophagy. As autophagy can be activated by rapamycin (and rapamycin inhibits long‐term memory), our aim was to test the hypothesis that autophagy inhibitors would enhance long‐term memory. To examine if learning alters autophagosome number, we used male reporter mice carrying the GFP‐LC3 transgene. Using these mice, we observed that training in the Morris water maze task increases the number of autophagosomes, a finding contrary to our expectations. For learning and memory studies, male Long Evans rats were used due to their relatively larger size (compared to mice), making it easier to perform intrahippocampal infusions in awake, moving animals. When the autophagy inhibitors 3‐methyladenine (3‐MA) or Spautin‐1 were administered bilaterally into the hippocampii prior to training in the Morris water maze task, the drugs did not alter learning. In contrast, when memory was tested 24 hours later by a probe trial, significant impairments were observed. In addition, intrahippocampal infusion of an autophagy activator peptide (TAT‐Beclin‐1) improved long‐term memory. These results indicate that autophagy is not necessary for learning, but is required for long‐term memory formation.     

Two-hour glucose and insulin responses after a standardized oral glucose load in relation to serum gamma-glutamyl transferase and alcohol consumption

Summary In a population study of 4,763 middle-aged men, the 120-min responses of blood glucose as well as plasma IRI in OGTTs were studied in subgroups of the screening population with different levels of gamma-glutamyl transferase (GGT) and/or defined alcohol consumption levels. In the group with low GGT (below the median, n=2, 196), both 120-min blood glucose and plasma IRI tended to be lower than in the whole screening cohort and there was a significantly smaller number of cases with 120-min blood glucose values 7.0 mmol/l. In a group of ideological alcohol abstainers the values of fasting as well as 120-min blood glucose were largely the same as in the average middle-aged men. In members of the study population with increased screening GGT, however, both fasting and, especially, 120-min values of blood glucose and plasma IRI were higher than in the average males, particularly in the cases in which the interview revealed chronic heavy alcohol consumption as the predominant underlying factor associated with elevated GGT. In these individuals, the prevalence of 120-min screening OGTT blood glucose values 7.0 mmol/l was 26%, in comparison with 13% in the average men and 9% in the individuals with screening GGT left of the median. This indicates that GGT and alcohol consumption are of clinical importance both for the results and interpretation of OGTTs.     

Attachment of Carbohydrate to the Variable Region of Myeloma Immunoglobulin Light Chains

Approximately 15 per cent of the light chains from homogeneous immunoglobulins in patients with multiple myeloma contain an oligosaccharide group. Five human myeloma light chains that contained carbohydrate were studied. The sequence Asn-[unk]-Ser/Thr was at the site of carbohydrate attachment in all light chains. The carbohydrate group was attached to the asparagine residue of this triplet sequence which in all five light chains was located in the variable region. The occasional occurrence of carbohydrate in myeloma light chains is seen as the consequence of a variable region mutation creating an Asn-[unk]-Ser/Thr sequence to which carbohydrate is attached by an enzyme capable of recognizing the characteristic triplet sequence.     

Characterization of D-3,4-cyclopropylglutamates as N-methyl-D-aspartate receptor agonists

The 4 configurational isomers of D-3,4-cyclopropylglutamate (D-CGA) have been synthesized and analyzed for their interactions as excitatory amino acid recognition sites. Additionally, functional assessment of the action of these compounds at the N-methyl-D-aspartate (NMDA) receptor was performed. All 4 analogs function as agonists at the NMDA receptor as evidenced by their ability to stimulate [3H]MK-801 binding to the coupled PCP recognition site. Furthermore, the rank order of potency of these compounds in stimulating [3H]MK-801 binding corresponds with their Ki values for the displacement of NMDA-selective L-[3H]glutamate and [3H]CGS-19755 binding (D-CGA-C greater than D-CGA-B greater than D-CGA-D greater than D-CGA-A). The D-CGA-C isomer has affinity and potency at the NMDA receptor similar to the endogenous agonist, L-glutamate. This high potency coupled with greater specificity than L-glutamate, makes D-CGA-C a potentially useful pharmacological tool for the study of this receptor.     

On the role of the transmembrane anchor sequence of influenza hemagglutinin in target cell recognition by class I MHC-restricted, hemagglutinin-specific cytolytic T lymphocytes

We have examined the requirement for the transmembrane hydrophobic anchor sequence of the influenza hemagglutinin (HA) in the formation of the antigenic moiety on the surface of target cells recognized by class I MHC-restricted murine CTL. For this analysis we have used a line of CV-1 monkey epithelial cells that express the transfected murine H-2Kd gene product as target cells and have used recombinant SV40-based late replacement vectors to achieve expression of genes encoding wild-type and mutant forms of HA. We have found that the majority of Kd-restricted HA-specific CTL clones recognize target cells that express a secreted HA molecule that lacks the transmembrane and cytoplasmic domains of the parent glycoprotein. Several Kd-restricted CTL clones that recognize subtype-specific and crossreactive epitopes on HA fail to recognize the anchor-negative, secreted HA or chimeric HA molecules containing the transmembrane and cytoplasmic domains of unrelated glycoproteins. These CTL clones appear to be directed to antigenic epitopes located within the transmembrane domain of HA, as defined by their capacity to recognize target cells sensitized with a synthetic 23-amino-acid peptide corresponding to sequences within this domain. The implications of these results for class I MHC-restricted CTL recognition are discussed.     

Renal revascularization in Takayasu arteritis-induced renal artery stenosis

PURPOSE: This study was undertaken to define the long-term effects of renal revascularization on blood pressure, and renal and cardiac function in patients with Takayasu arteritis-induced renal artery stenosis (TARAS). METHODS: Twenty-seven patients (25 women; mean age, 27 years) with TARAS underwent intervention. Primary, primary assisted, and secondary patency rates were determined, and the late effects on blood pressure, renal and cardiac function, and survival were analyzed. RESULTS: All patients had hypertension (mean blood pressure, 167/99 mm Hg; 2.5 antihypertensive medications per patient). Mean estimated glomerular filtration rate in patients not receiving hemodialysis was 76 mL/min, and in five patients serum creatinine concentration was greater than 1.5 mg/dL. Three patients were hemodialysis-dependent, and two had intractable congestive heart failure. Forty interventions were performed, including 32 aortorenal bypass procedures, two repeat implantations, four nephrectomies, and two transluminal angioplasty procedures. Postoperative morbidity was 19%. There were no deaths. During follow-up (mean, 68 months), three graft stenoses, all due to intimal hyperplasia, and three graft occlusions occurred. Two of three graft stenoses were successfully revised. At 1, 3, and 5 years of follow-up, primary patency was 87%, 79%, and 79%, respectively; primary assisted patency was 93%, 89%, 89%, respectively; and secondary patency was 93%, 89%, and 89%, respectively. Intervention resulted in a decrease in blood pressure to a mean of 132/79 mm Hg (P<.0001), and the need for antihypertensive medications was reduced to one per patient (P<.01). Mean glomerular filtration rate increased to 88 mL/min (P<.005), and two patients no longer required hemodialysis. Congestive heart failure resolved in both patients, and did not recur. There were three deaths during follow-up, with 5-year and 10-year actuarial survival of 96% and 80%, respectively. CONCLUSIONS: Renal revascularization to treat TARAS is durable, has a salutary effect on blood pressure, and enhances long-term renal and cardiac function. This response establishes renal revascularization as a successful and durable intervention for TARAS, and a benchmark to which other therapies should be compared.     

Spinal versus Epidural Anesthesia for Cesarean Section in Severely Preeclamptic Patients: A Retrospective Survey

Background: Selection of spinal anesthesia for severely preeclamptic patients requiring cesarean section is controversial. Significant maternal hypotension is believed to be more likely with spinal compared with epidural anesthesia. The purpose of this study was to assess, in a large retrospective clinical series, the blood pressure effects of spinal and epidural anesthesia in severely preeclamptic patients requiring cesarean section.

Methods: The computerized medical records database was reviewed for all preeclamptic patients having cesarean section between January 1, 1989 and December 31, 1996. All nonlaboring severely preeclamptic patients receiving either spinal or epidural anesthesia for cesarean section were included for analysis. The lowest recorded blood pressures were compared for the 20-min period before induction of regional anesthesia, the period from induction of regional anesthesia to delivery, and the period from delivery to the end of operation.

Results: Study groups included 103 women receiving spinal anesthesia and 35 receiving epidural anesthesia. Changes in the lowest mean blood pressure were similar after epidural or spinal anesthesia. Intraoperative ephedrine use was similar for both groups. Intraoperative crystalloid administration was statistically greater for patients receiving spinal versus epidural anesthesia (1780 +/- 838 vs. 1359 +/- 674 ml, respectively). Neonatal Apgar scores and incidence of maternal intensive care unit admission or postoperative pulmonary edema were also similar.