Renal artery stenosis caused by nonspecific arteritis (Takayasu disease): results of treatment with percutaneous transluminal angioplasty.

Renovascular hypertension is common in nonspecific aortoarteritis (Takayasu disease). The utility of percutaneous transluminal renal angioplasty in managing this disease has been reported infrequently, and technical problems in using this treatment method have not been described. We retrospectively evaluated the results of renal angioplasty in treating 33 stenoses in 20 patients. Each patient's diagnosis was based on the criteria established by the Aortitis Syndrome Research Committee of Japan. Criteria for selection of patients for angioplasty were (1) severe hypertension uncontrolled by single-drug therapy, (2) angiographic evidence of at least 70% stenosis of the renal artery with a pressure gradient of more than 20 mm Hg, and (3) a normal sedimentation rate. The transfemoral route was used to treat all 33 stenoses. Follow-up examinations included blood pressure and medication evaluation 1 day, 1 week, and 4-6 weeks after treatment, and thereafter at 6-month intervals. Technical success was obtained in 28 lesions (85%) in 17 patients (85%). All failures occurred in the presence of coexistent abdominal aortic disease and tight, proximal stenosis of the renal artery. Technical difficulties were attributed to the tough, noncompliant nature of the stenoses, which were difficult to cross and resisted repeated, prolonged balloon inflations. These patients experienced backache and a fall in systemic blood pressure during balloon inflation. In one patient, the ipsilateral renal vein was injured during angioplasty and required surgery. Clinical success was obtained in 14 (82%) of the 17 patients in whom technical success was achieved and included cure in six patients and improvement in eight others. Follow-up 1-18 months (mean, 8 months) after treatment showed restenosis in six (21%) of 28 lesions. We conclude that renal angioplasty in nonspecific arteritis is associated with technical difficulties; however, the short-term results are good and the complication rate is acceptable.


A combination of electrocardiographic methods represents a further step toward the noninvasive identification of patients with syndrome X.

Identification of patients with angina but normal coronary arteriograms (syndrome X) using noninvasive means would be desirable. The ability of four established exercise electrocardiographic methods to identify angina patients with and without coronary artery disease was compared with that of a method based on a combination of the above (combined method). A treadmill score, a multivariate method, the ST segment recovery loop, the ST/heart rate adjustment, and the combined method were applied to 112 patients who had typical exertional angina and positive exercise tests (greater than 1 mm ST segment depression); 90 had documented coronary artery disease and 22 had syndrome X. The combined method and the treadmill score had a significantly higher diagnostic accuracy (both 81%, as 91 of the 112 patients were correctly identified by both methods) than the multivariate (66%) and ST segment recovery loop (64%) methods (p less than 0.05). The ST/heart rate adjustment had a lower sensitivity for syndrome X than any other method (1 of 22). Thus methods that involve the assessment of both ST and non ST segment variables have greater accuracy in separating syndrome X and coronary artery disease patients than methods relying more heavily on ST segment changes.


Mechanisms of ring chromosome formation in 11 cases of human ring chromosome 21.

We studied the mechanism of ring chromosome 21 (r(21)) formation in 13 patients (11 unique r(21)s), consisting of 7 from five families with familial r(21) and 6 with de novo r(21). The copy number of chromosome 21 sequences in the rings of these patients was determined by quantitative dosage analyses for 13 loci on 21q. Nine of 11 r(21)s, including the 5 familial r(21)s, showed no evidence for duplication of 21q sequences but did show molecular evidence of partial deletion of 21q. These data were consistent with the breakage and reunion of short- and long-arm regions to form the r(21), resulting in deletion of varying amounts of 21q22.1 to 21qter. The data from one individual who had a Down syndrome phenotype were consistent with asymmetric breakage and reunion of 21q sequences from an intermediate isochromosome or Robertsonian translocation chromosome as reported by Wong et al. Another patient, who also exhibited Down syndrome, showed evidence of a third mechanism of ring formation. The likely initial event was breakage and reunion of the short and long arms, resulting in a small r(21), followed by a sister-chromatid exchange resulting in a double-sized and symmetrically dicentric r(21). The phenotype of patients correlated well with the extent of deletion or duplication of chromosome 21 sequences. These data demonstrate three mechanisms of r(21) formation and show that the phenotype of r(21) patients varies with the extent of chromosome 21 monosomy or trisomy.


Identification of novel RFLPs in the vicinity of CpG islands in Xq28: application to the analysis of the pattern of X chromosome inactivation.

Probes for CpG islands were cloned from the distal long arm of the human X chromosome; three of them were found to be polymorphic. A HindIII RFLP was identified by the probe 2-25 (DXS606), and it was mapped to the Xq27-Xq28 boundary. Probes 2-19 (DXS605) and 2-55 (DXS707), which identify EcoRI and MspI polymorphisms, respectively, have been mapped to the distal part of Xq28, in the G6PD-RCP/GCP gene region. Probe 2-19 has been further localized about 16 kb from the 3' end of the G6PD gene. The new RFLPs may be useful for the precise mapping of the many disease genes localized in this part of the human X chromosome. Probe 2-19 is highly informative, and it has been studied in greater detail. Using the methylation-sensitive rare-cutter enzyme EagI in conjunction with the polymorphic EcoRI site, we were able to demonstrate that the RFLP may be used both to study randomness of X chromosome inactivation and for carrier detection in X-linked syndromes where nonrandom X inactivation occurs. It is conceivable that the combined use of 2-19 and of the probes described so far (pSPT-PGK and M27 beta) will make analysis of X inactivation feasible in virtually every female.


Parental origin of factor IX gene mutations, and their distribution in the gene.

Genomic amplification followed by direct sequencing enabled us to establish the causative mutation in 67 unrelated hemophilia B patients of predominantly German origin. With the detection of the mutation, extensive pedigree analysis has become feasible. We therefore anticipated that determination of the origin of mutation could be achieved in a comparatively great number of families. Although these investigations often were restricted by the availability of blood samples from the maternal grandparents or great-grandparents, we were able to prove a de novo mutation in 9 of 20 families with sporadic hemophilia B and in 3 of 20 families with a history of the disease. This could be achieved with the aid of RFLP analysis and, in one case, where the mutation is still unknown, with the aid of biochemical and immunological factor IX assays. Since the maternal grandfather was decreased in two of these families, the germ line of origin could not be determined precisely. In the remaining families, the female and male germ lines turned out to be the origin of mutation in six and four cases, respectively, and an effect of paternal age on the mutations observed could not be excluded. Furthermore, our data indicate that the hemophilia B gene pool is mainly renewed by variable mutations.


Microdeletions of chromosome 17p13 as a cause of isolated lissencephaly.

Lissencephaly (agyria-pachygyria) is a brain malformation manifested by a smooth cerebral surface, resulting from arrest of neuronal migration at 10-14 wk gestation. Type I, or classical, lissencephaly can occur either in association with the Miller-Dieker syndrome (MDS) or as an isolated finding, termed "isolated lissencephaly sequence" (ILS). About 90% of MDS patients have visible or submicroscopic deletions of 17p13.3. We therefore investigated the possibility that some ILS patients have smaller deletions in this chromosomal region. Forty-five ILS patients with gyral abnormalities ranging from complete agyria to mixed agyria/pachygyria and complete pachygyria were studied. RFLP analysis with five polymorphic loci in 17p13.3 was performed on all patients and their parents. Somatic cell hybrids were constructed on three patients, to confirm a deletion or to determine the boundaries of a deletion. In-situ hybridization using cosmid probes from within a newly defined lissencephaly critical region was performed on 31 patients as a further method of deletion detection. Six submicroscopic deletions were detected (13.3%). Three of the deletions among 45 ILS patients were detected by RFLP analysis, 4 deletions in 31 patients were detected by in situ hybridization, and one deletion was detected only by somatic cell hybrid studies (in situ hybridization was not performed). Overall, in situ hybridization proved to be the most rapid and sensitive method of deletion detection. The centromeric boundary of these deletions overlapped that of MDS patients, while the telomeric boundary for four ILS deletions was proximal to that of MDS and narrows the critical region for a lissencephaly locus.(ABSTRACT TRUNCATED AT 250 WORDS)


Mapping of 262 DNA markers into 24 intervals on human chromosome 11.

We have extended our mapping effort on human chromosome 11 to encompass a total of 262 DNA markers, which have been mapped into 24 intervals on chromosome 11; 123 of the markers reveal RFLPs. These clones are scattered throughout the chromosome, although some clustering occurs in R-positive bands (p15.1, p11.2, q13, and q23.3). Fifty-two of the markers were found to contain DNA sequences conserved in Chinese hamster, and some of these 52 also cross-hybridized with DNA from other mammals and/or chicken. As the length of chromosome 11 is estimated at nearly 130 cM, the average distance between RFLP markers is roughly 1 cM. The large panel of DNA markers on our map should contribute to investigations of hereditary diseases on this chromosome, and it will also provide reagents for constructing either fine-scale linkage and physical maps or contig maps of cosmids or yeast artificial chromosomes.


Isolation and mapping of 68 RFLP markers on human chromosome 6.

We have isolated 68 new RFLP markers on human chromosome 6. Of these, 64 were localized on chromosomal bands by the fluorescent in-situ hybridization (FISH) method, 25 on the short arm and 39 on the long arm. Their distribution was uneven; the markers were localized predominantly in regions of R-positive banding. Eleven markers defined VNTR loci. This expanded collection of DNA markers will contribute to high-resolution linkage mapping of genes causing inherited disorders and will provide useful reagents for isolation of putative tumor-suppressor genes on chromosome 6 that appear to be involved in malignancies. Furthermore, the new markers will be guideposts for detailed linkage and physical maps of this chromosome.


Patterns of association between genetic variability in apolipoprotein (apo) B, apo AI-CIII-AIV, and cholesterol ester transfer protein gene regions and quantitative variation in lipid and lipoprotein traits: influence of gender and exogenous hormones.

Patterns of RFLP association were studied, to identify gene regions influencing quantitative variation in lipid and lipoprotein traits (coronary artery disease [CAD] risk factors or metabolically related traits). Subjects (118 female and 229 male; age 20-59 years) were selected for health. Multiple RFLPs were used to sample variability in regions around genes for apolipoprotein (apo) B (restriction enzymes HincII, PvuII, EcoRI, and XbaI), apo AI-CIII-AIV (BamHI, XmnI, TaqI, PstI, SstI, and PvuII) and cholesterol ester transfer protein (TaqI). Separate analyses were done by gender. The sample was truncated at mean +/- 4 SD, to remove extreme outliers. There was no significant gender difference in RFLP genotype frequency distribution. After trait-level adjustment to maximize removal of concomitant variability, analysis of variance was used to estimate the percentage trait phenotypic variance explained by measured variability in the gene regions studied. Fewer gene regions were involved in men, with less influence on quantitative trait variation than in women, in whom hormone use affected association patterns. Gender differences imply that pooling genders or adjusting data for gender effects removes genetic information and should be avoided. The association patterns show that variability around the candidate genes modulates trait levels: the genes are contributors to the genetics of CAD risk variables in a healthy sample.


Treatment of stress response during balanced anesthesia. Comparative effects of isoflurane, alfentanil, and trimethaphan.

Acute hypertensive responses during nitrous oxide-opioid-relaxant anesthesia are a common clinical problem. In adult men undergoing radical prostatectomy procedures and anesthetized with a standardized technique, we evaluated the effectiveness of alfentanil, isoflurane, and trimethaphan in treating acute hemodynamic and stress hormone responses to surgical stimulation. Stress hormone concentrations were measured 1 min before skin incision, after the onset of an acute hypertensive response, and after returning the mean arterial pressure to within 10% of the preincision values with one of the three treatment modalities. Pretreatment plasma alfentanil concentrations (151 +/- 47 to 156 +/- 47 ng.ml-1) and end-tidal nitrous oxide concentrations (66 +/- 2 to 68 +/- 2%) were similar in all three groups. Acute hypertensive events were associated with significantly increased concentrations of catecholamines and vasopressin (antidiuretic hormone [ADH]). Whereas intravenous alfentanil returned all hormone concentrations to preincision values, norepinephrine and glucose concentrations were significantly increased after adjunctive isoflurane administration. Although trimethaphan decreased the norepinephrine concentration, the epinephrine, beta-endorphin, cortisol, ADH, and glucose concentrations were significantly increased compared to preincision values. However, the persistent elevation in the posttreatment ADH concentration in the trimethaphan group was the only significant difference between the three groups. Mean (+/- standard deviation) times to awakening (2.8 +/- 3.3 to 3.8 +/- 4.2 min), extubation (8.1 +/- 4.8 to 10.3 +/- 8.5 min), and orientation (19.6 +/- 20.4 to 24.6 +/- 19.1 min) were similar in all three groups. Naloxone was required more frequently in patients in the alfentanil (35%) and isoflurane (24%) groups than in the trimethaphan group (4%).(ABSTRACT TRUNCATED AT 250 WORDS)


[Positional cloning of genes responsible for hereditary tumors].

Recently, remarkable progress in molecular biology has enabled isolation of genes responsible for hereditary tumors such as retinoblastoma (RB), Wilms' tumor (WT), von Recklinghausen neurofibromatosis (NF 1), and familial adenomatous polyposis (FAP). Since patients with FAP develop multiple adenomatous polyps in the colon, some of which progress to colon cancer, isolation of the FAP gene allows us a rare opportunity to study genetic events underlying the well defined morphological changes during progression of colorectal tumors. In this report, we presented an approach called "positional cloning" which has become a powerful tool for identifying genes responsible for hereditary tumors, as well as characteristics of some of such genes.


[An overview of new prognostic factors in lung cancer].

The knowledge of prognostic factors such as TNM, performance status, and sex is essential for predicting patient outcome and optimal trial design and analysis. Recent advances in cytogenetics and molecular biology have yielded new prognostic factors such as DNA ploidy, oncogenes and oncogene product. New prognostic factors can predict patient outcome and should be incorporated for the multivariate analysis of prognostic factors. They can provide a guideline for selecting special patient population suitable for adjuvant chemotherapy even in the early stage of lung cancer.


[New prognostic factors in human gastric carcinomas].

Correlation between the expression of growth factor/receptor systems or the alterations of tumor suppressor genes and biological malignancy of gastric cancer was described. Overexpression of many growth factors/receptors, such as EGF, TGF alpha, EGF receptor and ERBB2, and reduction of type I receptor for TGF beta may be linked with new prognostic factors of gastric carcinomas. The expression of cripto, a novel gene of EGF family, shows a tendency to correlate with tumor staging of well differentiated gastric adenocarcinomas. p53 gene abnormalities take place in 60% of gastric carcinomas including early stage carcinoma. Loss of heterozygosity on chromosomes 1q, 7p and 7q is frequently observed in advanced gastric carcinomas of well differentiated type. Molecules which regulate tumor invasion and metastasis such as nm23, tissue inhibitor of metalloproteinase (TIMP) and endogenous galactoside-binding lectin may provide for prognostic factors of gastric cancer.


[Expression of c-erbB-2 protein and vessel invasion in colorectal cancer].

Using sections of formalin-fixed, paraffin-embedded tissues from 64 colorectal cancer patients, the expression of c-erbB-2 oncoprotein was studied immunohistochemically. Twenty-seven percent of the cases with liver metastasis showed positive staining. On the other hand, only 3% of cases without liver metastasis were positive. Expression rates of c-erbB-2 protein in liver metastasis cases showed no significant difference between primary operation (26%) and recurrence (27%). Of all c-erbB-2 positive patients, 90% (9/10) had liver metastasis. Secondly, vessel invasions of 45 rectal cancer patients were studied using Victoria Blue (VB) elastic staining and endothelial staining by factor VIII-related antigen and Ulex europaeus agglutinin I (UEA-I) lectin. VB-HE double stain was efficacious to detect vascular invasion, but endothelial staining was not. There were statistically more vascular invasions in 30 patients with liver or lymph node metastases than in those without metastasis. And in cases with metastasis, many vascular invasions into the extra-muscular layer were seen. Both vascular invasions and c-erbB-2 protein were valuable indicators of possible liver metastasis.


Identification of Hantaan virus-related structures in kidneys of cadavers with haemorrhagic fever with renal syndrome.

The etiologic agent of haemorrhagic fever with renal syndrome (HFRS), Hantaan virus, was first isolated in 1976. Since then numerous Hantaan-like viruses have been isolated and five serotypes of Hantavirus have been recognized. Serological studies indicate that these viruses are globally distributed, with each serotype occurring in specific areas. Hantaan virus has been intensively studied antigenically, biochemically, and genetically. However there is still a paucity of information on the pathogenesis of Hantaan virus in the human host. In this paper, we report the detection by thin section immune electron microscopy of the occurrence of numerous dense precipitates, typical inclusion bodies, a surface antigen layer, as well as Hantaan virion-like structures in the kidneys of patients that died during the acute phase of HFRS. These findings may shed some light on understanding the pathogenesis of HFRS in target organs most affected by the disease, such as the kidneys.


5-hydroxytryptamine is a fast excitatory transmitter at 5-HT3 receptors in rat amygdala.

A fast excitatory synaptic potential mediated by 5-hydroxytryptamine (5-HT) was recorded in rat lateral amygdala neurons in brain slices. The synaptic potential has brief duration (tens of milliseconds), is mimicked by 5-HT, is potentiated by a 5-HT uptake inhibitor, and is blocked by selective 5-HT3 receptor antagonists. The underlying synaptic current reversed polarity at about 0 mV. This is an example of fast neurotransmission in the mammalian brain mediated by an amine rather than an amino acid. The antiemetic, anxiolytic, and perhaps antipsychotic actions of 5-HT3 antagonists might result from blockade of such synapses.


Pertussis toxin and voltage dependence distinguish multiple pathways modulating calcium channels of rat sympathetic neurons.

Agonist-induced suppression of current in voltage-gated Ca2+ channels was studied in rat sympathetic neurons. We have previously distinguished two intracellular signaling pathways used by muscarinic agonists to suppress neuronal Ca2+ current-one fast and membrane delimited, the other slow and acting via a diffusible second messenger. We now show that the fast pathway is sensitive mainly to pertussis toxin and shifts the gating of Ca2+ channels to more positive voltages (voltage dependent). The slow pathway is pertussis toxin insensitive and depresses currents at all test potentials (voltage independent). Muscarinic agonists may also activate a pertussis toxin-insensitive fast pathway. alpha-Adrenergic agonists use the fast pertussis toxin-sensitive and the fast insensitive pathways, but not the slow one.


Probing the active site of the reconstituted aspartate/glutamate carrier from bovine heart mitochondria: carbodiimide-catalyzed acylation of a functional lysine residue.

Upon modification of the reconstituted aspartate/glutamate carrier by various amino acid-reactive chemicals a functional lysine residue at the exofacial binding site was identified. The inactivation of transport function by the lysine-specific reagents pyridoxal phosphate (PLP, IC50 400 microM) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS, IC50 300 microM) could specifically be suppressed by the substrates aspartate and glutamate; a 50% substrate protection was observed at half-saturation of the external binding site. The same held true for 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, IC50 500 microM) and diethyl pyrocarbonate (DEPC, IC50 20 microM), two reagents known to modify carboxylic or histidinyl side-chains, respectively. EDC, however, turned out to catalyze an acylation of the active site lysine by activating carboxyls that had to be present in the incubation medium. This special mechanism, which was proven by protein labelling using EDC/[14C]succinate, necessitates a lysine side-chain of high reactivity and low pK, since the modification had to occur at pH less than or equal to 6.5, i.e. not too far from the pK of the carboxyl to be activated. All reagents applied, additionally including 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS, IC50 10 microM), were effective at this pH. Competition experiments revealed interaction of EDC, PLP, SITS and probably DIDS at the same active site lysine. For DEPC a lysine modification could not be ruled out. Yet, a model comprising a histidine juxtaposed to the lysine seems to be appropriate.


Comparison of the conversion rates of alpha-linolenic acid (18:3(n - 3)) and stearidonic acid (18:4(n - 3)) to longer polyunsaturated fatty acids in rats.

The delta 6-desaturase reaction is regarded to be the rate-limiting step in the conversion of linoleic acid (18:2(n - 6)) to arachidonic acid (20:4(n - 6)). The same is probably also the case with the conversion of alpha-linolenic acid (18:3(n - 3)) to eicosapentaenoic acid (20:5(n - 3)). However, there are very few in vivo studies that directly compared the conversion rate between 18:3(n - 3) and stearidonic acid (18:4(n - 3)), which is the delta 6-desaturated product of 18:3(n - 3). We compared this rate by feeding rats on a lipid-free diet supplemented with lard (9%, w/w) and 18:3(n - 3) ethyl ester (1%) diet or on a diet containing lard (9%) and 18:4(n - 3) ethyl ester (1%). A lard (10%)-supplemented diet was used as the control diet. The fatty acid compositions of total phospholipids, triglycerides and free fatty acids of both liver and plasma were measured after 1 or 3 weeks on different diets. The molar ratio of 20:5(n - 3) of most lipid fractions was about 2-fold higher in rats fed the 18:4(n - 3)-supplemented diet than in rats fed the 18:3(n - 3)-supplemented diet. 18:4(n - 3) was found in the liver lipid fraction in only a very small amount, even in the 18:4(n - 3)-supplemented groups. Thus, desaturation at C-6 is suggested to be the rate-limiting step in the conversion of 18:3(n - 3) to 20:5(n - 3).


Induction by psychotropic drugs and local anesthetics of DnaK and GroEL proteins in Escherichia coli.

We examined effects of psychotropic drugs and local anesthetics on the synthesis of heat shock proteins in Escherichia coli. Chlorpromazine, a phenothiazine derivative, was shown to induce DnaK and GroEL proteins, major heat shock proteins in E. coli. The inductions of these proteins were not observed in an rpoH (= htpR) amber mutant strain, indicating that the heat shock sigma factor sigma 32 was required for their inductions. Northern blot hybridization analysis revealed that chlorpromazine induced increases of messenger RNAs for the DnaK and GroEL proteins. Thus, the induction occurred at the level of transcription. Chlorpromazine also induced non-heat shock proteins with molecular masses of 21 kDa, 20 kDa, and 17 kDa, even in the rpoH mutant strain. Other psychotropic drugs and local anesthetics, namely, dibucaine, lidocaine, imipramine, tetracaine and procaine, also induced DnaK and GroEL proteins and the small molecular weight proteins.


Lipoprotein-associated coagulation inhibitor (LACI) is a cofactor for heparin: synergistic anticoagulant action between LACI and sulfated polysaccharides.

Lipoprotein-associated coagulation inhibitor (LACI) is a plasma-derived protein that inhibits tissue factor (TF)/factor VIIa-induced coagulation in a factor Xa-dependent manner. The roles of endogenous plasma LACI and exogenously added LACI and heparin, in the regulation of coagulation, initiated via the intrinsic and extrinsic pathways, were studied using the activated partial thromboplastin time (APTT) and the modified prothrombin time (PT) assays, respectively. Both LACI-depleted plasma and normal plasma have identical APTTs and similar prolongations of the APTT in response to heparin; both are fully anticoagulated (arbitrarily defined as clotting times of greater than 1 hour) at similar concentrations of heparin. These results indicate that heparin is an effective anticoagulant when coagulation is initiated by the intrinsic pathway and that endogenous LACI is not significantly involved in the regulation of this pathway. The PT of normal plasma is only marginally longer than that of LACI-depleted plasma in the absence of heparin, suggesting that endogenous plasma LACI has a very limited capacity to inhibit TF-induced clotting. However, in the presence of heparin, the PTs of LACI-depleted plasma and normal plasma are different. Prolongation of the PT occurred only moderately and linearly with increasing concentrations of heparin in LACI-depleted plasma. In contrast, normal plasma showed a greater extent of PT prolongation in response to heparin and the plasma became fully anticoagulated at a certain threshold concentration of heparin. These results suggest that LACI serves as a cofactor for heparin and thus greatly enhances the inhibition of TF-induced coagulation. LACI-depleted plasma was supplemented with purified recombinant LACI and/or heparin and the effects on TF-induced clotting were studied. A combination of LACI and heparin greatly enhanced anticoagulation compared with LACI or heparin alone. Many sulfated polysaccharides were also found to enhance the LACI-dependent inhibition of TF-induced clotting. By weight, the relative potencies of these compounds are: low molecular weight heparin (mean Mr, 5,100) greater than unfractionated heparin greater than low molecular weight heparin (mean Mr, 3,700) greater than pentosan polysulfate greater than dermatan sulfate greater than dextran sulfate greater than heparan sulfate. Based on the above results, it is concluded that LACI is a cofactor for heparin in the inhibition of TF-induced clotting and that LACI and sulfated polysaccharides act synergistically in whole plasma.


Treatment of advanced pancreatic carcinoma with the somatostatin analogue BIM 23014. Preliminary results of a pilot study.

Treatment of advanced pancreatic cancer has not improved substantially in recent years. The search for new agents or new therapeutic modalities may be critical for further development in the therapy of this disease. Experimental and clinical findings suggest that it might be possible to develop a new hormonal therapy for exocrine cancer of the pancreas based on new somatostatin analogues. Preliminary results indicate clinical activity and increased survival in some patients. In this study, 19 patients with advanced exocrine pancreatic carcinoma were given the somatostatin analogue BIM 23014 using a range of doses from 250 micrograms/day to 1 mg/day. One patient had a partial response, 6 patients had stable disease, and 11 had progressive disease. Six patients showed a sharp improvement in pain and performance status. Side effects were mild. Plasma levels of growth hormone were evaluated in ten patients and remained unchanged. The clinical activity observed, even if limited, warrants further investigation using more appropriate schedules and administration techniques.


Gestational and nongestational trophoblastic tumors distinguished by DNA analysis.

In three patients in whom a diagnosis of gestational trophoblastic tumor was possible on the basis of pathology and elevated levels of serum human chorionic gonadotrophin, locus-specific minisatellite probes were used to identify restriction fragment length polymorphisms (RFLP) in DNA from the tumor, the patient, and her partner. On the basis of results from these studies, one tumor, originally diagnosed as a germ cell tumor, was reclassified as a gestational choriocarcinoma, whereas a second tumor, diagnosed as gestational choriocarcinoma, was shown to be of nongestational origin. In the third case, a diagnosis of gestational trophoblastic tumor was confirmed, but in this case the androgenetic origin of the tumor indicated that it was derived, not from the antecedent term pregnancy, but from a previous pregnancy with hydatidiform mole. This study clearly demonstrates the value of DNA analysis in the classification of tumors with trophoblastic differentiation.


Aberrant expression of the c-erbB-2/neu protooncogene in ovarian cancer.

Overexpression of the c-erbB-2/neu protooncogene has recently been shown in ovarian tumors collected from the United States. It is known that environmental and cultural factors may contribute to certain types of cancer, therefore, we examined expression of c-erbB-2/neu in ovarian tumors collected from China by immunohistochemical staining. Out of 81 tumor specimens, 57 (70.4%) were found to be immunopositive, whereas only one out of 17 (5.9%) normal ovarian tissue samples was slightly positive. Our results indicate that overexpression of c-erbB-2/neu is a general phenomenon for ovarian cancer regardless of different population. To search for a c-erbB/neu overexpressing cell line for future study on molecular mechanism, we also analyzed 13 cancer cell lines from the female genital tract for expression of c-erbB-2/neu. The c-erbB-2/neu RNA was found to be overexpressed at least 100-fold in one of the four ovarian cancer cell lines examined. An aberrant c-erbB-2/neu RNA was also found to be overexpressed in this cell line. Southern blot analysis indicated that the c-erbB-2/neu was amplified 2-4-fold in this line, and some of these alleles have structural alteration which may account for expression of the aberrant c-erbB-2/neu RNA. Since the 2-4-fold gene amplification is not proportional to the greater than 100-fold overexpression in RNA, other mechanisms such as transcriptional or posttranscriptional control must be involved in overexpression of this gene in ovarian cancer.


TNF-alpha production by U937 promonocytes is enhanced by factors released from HIV-infected T4 lymphocytes: TNF-alpha is one of the mediators causing lysis of HIV-infected T4 cells.

In the present study, we have shown that the addition of culture supernatants from HIV-infected SupT1 cells (T4) but not from noninfected cells markedly increased the production of TNF-alpha by U937 promonocytic cells after stimulation with phorbol 12-myristate 13-acetate (PMA). Pretreatment of supernatants with the antibodies to granulocyte/macrophage colony-stimulating factor (GM-CSF) or TNF-alpha, but not interferon-gamma, significantly diminished this enhancing effect. These results suggest that HIV may play an indirect role by producing cytokines from infected T4 cells that can lead to an increased production of TNF-alpha by monocytic cells. Further, TNF-alpha produced by U937 cells following stimulation with PMA plus lipopolysaccharide or with phytohemagglutinin induced lysis of HIV-infected T cells. TNF-alpha-induced cytotoxicity was markedly higher toward HIV-infected than toward noninfected T4 cells. Addition of antibody to TNF-alpha during the cytotoxic phase of response resulted in a reduction of about 50% in the percentage of cytotoxicity, indicating TNF-alpha as one of the lytic mediators.


Baculovirus-mediated expression and characterisation of rat glycogen synthase kinase-3 beta, the mammalian homologue of the Drosophila melanogaster zeste-white 3sgg homeotic gene product.

Molecular cloning of glycogen synthase kinase-3 (GSK-3) has demonstrated the existence of a novel form, termed GSK-3 beta, which is highly related to the well characterised GSK-3 alpha protein but derived from a distinct gene. The cDNA cloning also revealed a striking degree of amino acid identity between the two GSK-3 proteins, particularly the beta-form, and the zeste-white3/shaggy (zw3sgg) homeotic gene of Drosophila melanogaster. Abrogation of zw3sgg causes pleiotropic effects on fruitfly development affecting segmental organisation and cell fate determination. In view of the potential importance of GSK-3 beta in mammalian development and the lack of previous characterisation, we have expressed this protein in insect cells using recombinant baculovirus. A rapid purification scheme has been developed yielding essentially pure GSK-3 beta protein in three chromatographic steps. The protein has autonomous protein kinase activity and similar, but not identical, substrate preferences to GSK-3 alpha. Both GSK-3 proteins activate the MgATP-dependent form of protein phosphatase-1 and thus display 'factor A' activity. Since GSK-3 beta exhibits an identical site specificity to GSK-3 alpha with respect to phosphorylation of the proto-oncogene/transcription factors c-jun and c-myc, it is likely that the Drosophila zw3sgg protein kinase has a similar specificity for such transcription factors which may underlie the pleiotropic phenotypes observed when the Drosophila homologue is mutationally inactivated.


Memory in helper T cells of minor histocompatibility antigens, revealed in vivo by alloimmunizations in combination with Thy-1 antigen.

A cooperative antibody response in which T helper (Th) cells recognize minor histocompatibility antigens (mha) and B cells recognize Thy-1 antigen, is used to explore memory in the T cell compartment. In contrast to B cell memory, Th memory reaches a plateau rapidly, although Th memory of Thy-1 itself (or an associated antigen) behaves exceptionally in this respect. The plateau then extends over several weeks at least. Single mha, among them H-Y, generate detectable memory. Incompatible H-2 antigens, including class I antigens on their own, inhibit this response through what appears to be a mechanism of intracellular antigenic competition. Antigen presentation in this system is by host cells, as judged by lack of donor-specific restriction. Memory resides in both the CD45RA+ and CD45RA- compartments, although the majority of memory Th cells have the latter phenotype.


Latent help to and from H-2 antigens.

An optimized system for probing allo-immunity to major histocompatibility complex (MHC) antigens by means of adoptive transfer is used to confirm and extend previous work showing that naturally occurring class I MHC antigens, while capable of inducing Th activity when presented in combination with other allo-antigens, fail to do so on their own. The Th activity which they do induce develops slowly, after repeated immunizations, and can properly be described as "latent". Latency, or "cripticity" as it is also termed, may help explain how autoimmune disease is initiated.


Restricted V alpha 2.3 gene usage by CD4+ T lymphocytes in bronchoalveolar lavage fluid from sarcoidosis patients correlates with HLA-DR3.

The alpha/beta T cell receptor (TcR) V gene usage of bronchoalveolar lavage (BAL) lymphocytes and peripheral blood lymphocytes (PBL) from 11 sarcoidosis patients and 4 healthy controls was investigated, using eight alpha/beta TcR V gene product-specific monoclonal antibodies (mAb). Twenty-seven percent (3/11) of the sarcoidosis patients had a highly significant increase in V alpha 2.3+CD4+ T lymphocytes in the bronchoalveolar space, while displaying normal frequencies of these T cells in peripheral blood. The reactivities with the remaining seven TcR mAb were normal. In the control group, no compartmentalization of any T cells was seen. Four of the patients expressed the HLA-DR3 (w17), DQw2 haplotype. Interestingly, the three patients with distinct signs of compartmentalized V alpha 2.3+CD4+ T cells all expressed this HLA haplotype. Additionally, a fourth patient with pronounced, although less significant, accumulation of V alpha 2.3+CD4+ T cells in the lung, was also HLA-DR3(w17), DQw2+. Expression of V alpha 2.3+CD4+ T cells in BAL of these patients correlated with clinical disease, as revealed on re-analyzing the four patients after 6 months or longer. Predominant TcR V alpha 2.3 gene usage in compartmentalized CD4+ BAL T lymphocytes, linked to HLA-DR3(w17), DQw2 haplotype, may thus indicate presence of a specific antigen localized to the lungs of sarcoidosis patients.


Intestinal intraepithelial lymphocyte T cells are resistant to lpr gene-induced T cell abnormalities.

The mucosal immune system of the gastrointestinal (GI) tract consists of Peyer's patches (PP), which are IgA inductive sites, and more diffuse effector regions which include cells in the intraepithelial lymphocyte (IEL) compartment. Since autoimmune MRL lpr/lpr (MRL/lpr) mice develop a proliferating CD3+, CD4-, CD8- (double negative; DN), B220+ T cell subset in systemic lymphoid tissue, we have initiated studies to determine the distribution of CD3+, DN, B220+ T cells (B220+ T cells or lpr/lpr T cells) in the GI immune system. Specifically, we examined T cell subsets separated according to expression of CD4, CD8, Thy-1, B220, alpha/beta T cell receptor (TcR) and gamma/delta TcR in PP and IEL of MRL/lpr mice at 6, 12 and 21 weeks of age. Increased numbers of CD3+ T cells were noted in both PP and spleen of 12- and 21-week-old mice in which the development of autoimmune disorders were also evident. However, normal numbers of CD3+ IEL T cells were seen in MRL/lpr mice in all three age groups tested. When the presence of T cell lymphadenopathy was examined in both IgA inductive and effector tissues, the PP followed the B220+ T cell pattern seen in the spleen, where approximately 30%-50% of CD3+ T cells in the PP of 12- and 21-week-old MRL/lpr mice expressed the phenotype of lpr/lpr T cells and greater than 90% were alpha/beta TcR+. On the other hand, B220+ T cells had not developed in PP or spleen of 6-week-old MRL/lpr mice. Of interest was the finding that IEL from lpr/lpr homozygous mice did not contain B220+ T cells in any age group tested. In this regard, the IEL of MRL/lpr mice comprised an identical pattern and frequency of CD4-/CD8+, CD4+/CD8-, DN and CD4+/CD8+ (double positive, DP) T cell subsets as their normal counterparts (i.e. MRL +/+, BALB/c and C3H/HeN mice) which consisted of approximately 75%, approximately 7.5%, approximately 7.5% and approximately 10%, respectively. Further, Thy-1, gamma/delta TcR and alpha/beta TcR expression in these four subsets of MRL/lpr IEL were very similar to normal mice. These results suggest that the intestinal IEL compartment is minimally affected by the lpr/lpr mutation which induces T cell abnormalities and indicate that B220+ T cells do not preferentially home to IEL. Further, our results support the concept that IEL T cells develop as a separate T cell lineage from thymus-derived cells.


Internalization of glycosyl-phosphatidylinositol (GPI)-anchored lymphocyte proteins. II. GPI-anchored and transmembrane molecules internalize through distinct pathways.

Ly-6A.2 (T cell-activating protein, TAP) and Thy-1 are glycosyl-phosphatidyl-inositol (GPI)-anchored proteins expressed on the surface of murine T lymphocytes. We have found that Ly-6A.2 (TAP) and Thy-1 are internalized by T cells. In the present study we have investigated whether these GPI-anchored proteins enter cells by endocytosis through coated pits. Two lines of evidence argue against the involvement of coated pits in the internalization of Ly-6A.2 (TAP) and Thy-1. First, drugs that effectively blocked the endocytosis of transferrin receptor and H-2 class I molecules, (which are known to be internalized via coated pits) did not inhibit the internalization of the GPI-anchored proteins. Second, in ultrastructural analyses, Ly-6A2 (TAP) and Thy-1, in contrast to the transferrin receptor, were rarely found in coated pits or vesicles. These observations suggest that the GPI-anchored proteins on T lymphocytes are internalized by a distinct pathway that does not involve endocytosis through coated pits.


Augmentation of major histocompatibility complex class I and ICAM-1 expression on glial cells following measles virus infection: evidence for the role of type-1 interferon.

An intracellular staining procedure for the cytoskeletal marker, glial fibrillary acidic protein of astrocytes, has been developed which allows flow cytometric phenotyping of astrocytes within complex mixtures of glial cells. Employing this technique, we show here that measles virus infection of rat mixed glial cell cultures results in a rapid augmentation of major histocompatibility complex (MHC) class I and ICAM-1 on the majority of astrocytes in culture. MHC class I levels are increased on macrophages/microglia but ICAM-1 expression is not normally affected on this cell type. Some MHC class II induction is also observed after virus infection but only on astrocytes. A type-I interferon (IFN)-inducible protein, Mx, was identified in cultured glial cells after infection. Qualitatively comparable MHC class I and ICAM-1 enhancement after addition of type-I IFN, supports the conclusion that this cytokine(s) released as a result of virus infection, is responsible for alterations in the expression of molecules on glial cells, that are involved in T cell recognition. Astrocytes after viral infection were more susceptible to alloantigen-specific cytotoxic T lymphocytes and cytotoxic T lymphocyte activity was substantially reduced in the presence of mAb specific for MHC class I, ICAM-1 and LFA-1 but not MHC class II. The relevance of these findings to T cell recognition of virus-infected cells in the central nervous system is discussed.


ICAM-1-independent lymphocyte transmigration across high endothelium: differential up-regulation by interferon gamma, tumor necrosis factor-alpha and interleukin 1 beta.

The adhesion of lymphocytes to cytokine-treated high endothelium was studied using cultured high endothelial cells (HEC). Pretreatment of the HEC layer with a variety of cytokines caused up-regulation of lymphocyte adhesion with the effects ordered interferon gamma (IFN-gamma) greater than tumor necrosis factor-alpha (TNF-alpha) greater than or equal to interleukin 1 beta (IL 1 beta). Increased lymphocyte adhesion was found to be independent of ICAM-1 as expression by HEC was not increased by cytokines and antibodies against ICAM-1 did not block adhesion. The peptide CS1 and anti-beta 1 integrin subunit antibodies, however, caused partial inhibition of lymphocyte adhesion thus indicating a role for fibronectin on HEC and alpha 4 beta 1 on lymphocytes. Study of the kinetics of lymphocyte adhesion showed that the effects of IFN-gamma and TNF-alpha were persistent and remained detectable 2.5 h after removal of the cytokines whereas the effects of IL 1 beta were transient and were not sustained beyond 1 h. All of the cytokines used caused transient increases in the number of surface-bound lymphocytes with IFN-gamma greater than TNF-alpha greater than or equal to IL 1 beta, however, the most dramatic effect was on the transmigration of lymphocytes across the HEC. Both IFN-gamma and TNF-alpha caused sustained increased transmigration with IFN-gamma having the greater effect. IL 1 beta had little effect on transmigration. This model demonstrates that the binding and transmigration of lymphocytes across HEC can be differentially regulated by the actions of individual cytokines. These results support the concept that locally produced cytokines regulate HEC function within the lymph node.


The genetics of IgG4 deficiency: role of the immunoglobulin heavy chain constant region and HLA loci.

IgG4 deficiency is very common (1/400 in the Italian population) and provides a good model for analyzing the genetic factors involved in Ig subclass deficiencies. We have previously reported an association between some immunoglobulin heavy chain constant region (IGHC) polymorphisms and the IgG4 deficiency. The associated polymorphisms spanned the region between the GP and the G4 genes. A larger sample composed of 50 healthy blood donors with IgG4 deficiency (less than 0.001 g/l IgG4), not carrying homozygous gene deletions, together with 82 first-degree relatives is now examined. The results confirmed the association of the deficiency with IGHC polymorphisms, and detected a new association with the HLA-D locus with a strong additive effect between the two systems. However, despite these associations and a highly significant risk for IgG4 deficiency within families, close linkage with either IGHC or HLA loci was not apparent by the affected sib pair method. These findings suggest that several concomitant, possibly cooperating, genetic factors may be involved in IgG4 deficiency.


CD4+ T cell-mediated killing of major histocompatibility complex class II-positive antigen-presenting cells (APC). III. CD4+ cytotoxic T cells induce apoptosis of APC.

A subset of CD4+ T cells, belonging to the T helper type 1 (Th1) cells, kills antigen-presenting cells (APC) in an antigen-specific and major histocompatibility (MHC) class II-restricted way. Evidence is presented that CD4+ cytotoxic T lymphocytes (CTL) induce apoptosis or programmed cell death within susceptible APC as witnessed by quantitative DNA fragmentation. Apoptosis is more reliable to determine cell death than the 51Cr-release assay, because some cells demonstrate resistance to CD4-mediated lysis in the 51Cr-release assay. Apoptosis becomes manifest after 2 to 4 h of incubation preceding the disintegration of the target cells which is detectable between 12 and 24 h as measured by the 51Cr-release assay. Unstimulated B cells, which are not killed, but function as APC, do not undergo apoptosis, whereas lipopolysaccharide or anti-mu-activated B cell blasts show apoptosis and are efficiently lysed. Several CD4+ Th2-type cells tested, which did not demonstrate killing of APC as measured by the 51Cr-release assay, are unable to mediate programmed cell death of appropriate APC. Actinomycin D or cycloheximide, inhibitors of transcription and translation, respectively, fail to prevent apoptosis of APC excluding the involvement of newly synthesized soluble products as mediators of killing. Pretreatment of CD4+ CTL, but not of APC with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, a specific inhibitor of the anion transport, efficiently prevents apoptosis of APC, although the secretion of interleukins is not affected. We propose, that upon contact of the CD4+ CTL with APC, molecules of yet undefined nature are activated and released in a polar fashion at the contact site and induce the endogenous pathway of programmed cell death.


Regulation of D-3 phosphoinositides during T cell activation via the T cell antigen receptor/CD3 complex and CD2 antigens.

An immediate consequence of T cell activation via the T cell receptor (TcR)/CD3 complex and CD2 antigen is the hydrolysis of phosphatidylinositol-(4,5)-bisphosphate and the generation of inositol-(1,4,5)-trisphosphate and diacylglycerol which then regulate intracellular calcium and protein kinase C. Changes in cellular levels of phosphoinositides phosphorylated on the D-4 and D-5 position during T cell activation have been well documented. Recently it has been proposed that phosphoinositides phosphorylated on the D-3 position of the inositol ring by a novel phosphoinositide (PI) 3 kinase may also be important in cell activation. In the present study we have examined the levels and regulation of D-3 phosphoinositides in T cells activated by the TcR/CD3 complex and CD2 antigens. The data show the existence of phosphatidylinositol-(3)-monophosphate [PtdIns(3)P], phosphatidylinositol-(3,4)-bisphosphate [PtdIns(3,4)P2] and phosphatidylinositol-(3,4,5)-trisphosphate [PtdIns(3,4,5)P3] in T cells. Activation of the TcR/CD3 complex or CD2 antigen results in modulation of PtdIns(3,4)P2 and a putative PtdIns(3,4,5)P3 in T cells but does not change levels of PtdIns(3)P. These data provide the first evidence that lipid products of a PI3 kinase exist in T cells.


Differential effect of interleukin 1 on naive and memory CD4+ T cells.

Freshly derived murine CD4+ T cells are divided into naive and memory cells based on the expression of CD45 isoforms. Cross-linking the T cell receptor CD3 complex either by plastic-bound anti-CD3 antibodies or the antibody presented on non-lymphoid Fc gamma receptor type II-positive Chinese hamster ovary cells in absence of competent antigen-presenting cells fails to activate naive cells to either secrete cytokines or to proliferate. In contrast, memory cells secrete their characteristic cytokines [interleukin (IL) 2, IL4, and interferon-gamma] and show significant proliferation to this stimulus. IL 1 however, is required for their optimal clonal expansion. Differential expression of IL 1 receptor mRNA in memory cells also correlate with their responsiveness to IL 1. Thus, these data reveal a basic difference in the requirements for activation of naive and memory CD4+ T cells.


Microtubule-associated protein autophosphorylation alters in vitro microtubule dynamic instability.

While phosphorylation of high-molecular-weight microtubule-associated proteins (MAPs) alters the assembly properties of microtubules in vitro, virtually nothing is known about the influence of MAP phosphorylation on the time-scale of microtubule polymer length redistribution. The latter has been used as an index of microtubule assembly/disassembly turnover as predicted by the dynamic instability model (Mitchison, T.M. and Kirschner, M.W. (1984) Nature 312, 237-242). We have now determined that under conditions leading to the incorporation of 8-10 mol phosphoryl groups per mol MAP-2 (and about 0.2 mol phosphoryl groups per mol MAP-1 and tau), we can reproducibly observe significant acceleration in the polymer length redistribution process in a manner consistent with greater microtubule dynamic instability. We have also found that MAP phosphorylation resulted in more extensive release of MAPs from microtubules as a function of increasing salt concentration. These results are consistent with a weakening of MAP-microtubule interactions upon phosphorylation.


Functional relationships between cyclodextrin glucanotransferase from an alkalophilic Bacillus and alpha-amylases. Site-directed mutagenesis of the conserved two Asp and one Glu residues.

Comparison of the amino acid sequences of cyclodextrin glucanotransferases (CGTases) with those of alpha-amylases revealed that two Asp and one Glu residues, which are considered to be the catalytic residues in alpha-amylases, were also conserved in CGTases. To analyze the function of the three conserved amino acid residues in CGTases, site-directed mutagenesis was carried out. The three mutant CGTases, in which Asp229, Glu257 and Asp328 were individually replaced by Asn or Gln, completely lost both their starch-degrading and beta-cyclodextrin-forming activities, whereas another mutant CGTase, in which Glu264 was replaced by Gln, retained these activities. The three inactive enzymes retained the ability to be bound to starch. These results suggest that Asp229, Glu257 and Asp328 play an important role in the enzymatic reaction catalyzed by CGTase and that a similar catalytic mechanism is present in both CGTases and alpha-amylases.


Acute ammonia toxicity is mediated by the NMDA type of glutamate receptors.

Previous experiments in our laboratory suggested that ammonium toxicity could be mediated by the NMDA type of glutamate receptors. To assess this hypothesis we tested if MK-801, a specific antagonist of the NMDA receptor, is able to prevent ammonium toxicity. Mice and rats were injected i.p. with 12 and 7 mmol/kg of ammonium acetate, respectively. 73% of the mice and 70% of the rats died. However, when the animals were injected i.p. with 2 mg/kg of MK-801, 15 min before ammonium injection, only 5% of the mice and 15% of the rats died. The remarkable protection afforded by MK-801 indicates that ammonia toxicity is mediated by the NMDA receptor.


Dual Bar homeo box genes of Drosophila required in two photoreceptor cells, R1 and R6, and primary pigment cells for normal eye development.

In the Bar mutation of Drosophila, ommatidial differentiation is known to be suppressed in the anterior portion of the eye. Our structural analysis shows that the Bar region contains a pair of homeo box genes, BarH1 and BarH2. These genes encode polypeptides similar in size and sequence and share a common homeo domain that is identical in sequence except for putative trans-activator-binding sites. We also show, by mosaic analysis and immunostaining with anti-BarH1/BarH2 antibodies, that BarH1 and BarH2 are not only specifically coexpressed but also functionally required in R1/R6 prephotoreceptors and primary pigment cells in developing ommatidia. In R1/R6, the expression of BarH1 and BarH2 appears to be regulated by rough and glass gene products. BarH1 and BarH2 proteins are essential to normal lens formation, formation of three types of pigment cells, and elimination of excess cells from mature ommatidia. Taken together, our results suggest that Bar homeo domain proteins may play key roles in the fate-determination processes of pigment cells and cone cells.


Effectiveness of advice to reduce alcohol consumption in hypertensive patients.

The relation between alcohol consumption and blood pressure is well recognized, and advice to reduce alcohol plays an important part in the management of hypertensive patients. We have evaluated the effectiveness of this advice in a randomized, controlled, single-blind clinical study. After a 2-week run-in period, hypertensive men regularly consuming more than 20 units/wk (1 unit = 10 g) of alcohol were randomly assigned either to the "advice" or control group and were seen at 2-week intervals over an 8-week study period. The outcome measures were: reported alcohol consumption (1-week retrospective diary), markers of alcohol consumption (serum gamma-glutamyl transpeptidase, aspartate aminotransferase, uric acid, mean corpuscular volume), and blood pressure (sitting and standing). Over 18 months, 67 men who drank more than 20 units/wk of alcohol were seen. Twenty-six either were excluded, refused to participate, or dropped out due to nonattendance. Forty-one patients completed the study. After intervention, reported alcohol consumption fell from 60 units/wk to around 30 units/wk in the advice group, whereas it remained between 50 and 60 units/wk in the control group (analysis of variance [ANOVA] F = 7.1, p less than 0.05). This was accompanied by falls in gamma-glutamyl transpeptidase (20.9%) and aspartate aminotransferase (18.1%), but no significant changes were seen in the control group. Standing diastolic blood pressure fell significantly in the advice group (from 101.5 mm Hg to 96.3 mm Hg) compared with the control group (ANOVA F = 4.8, p less than 0.05). The results suggest that advice to reduce alcohol consumption is a useful form of treatment for hypertensive patients who drink excessively.


Effects of antihypertensive therapy on insulin resistance.

In view of the likely prohypertensive effects of hyperinsulinemia and the presence of insulin resistance in primary hypertension, the effects of various antihypertensive therapies on insulin sensitivity need to be identified. The evidence strongly supports major beneficial effects of weight reduction and aerobic exercise. Deleterious effects have been shown for diuretics and most beta-blockers, whereas probable beneficial effects have been seen with alpha-blockers, one angiotensin converting enzyme inhibitor, and various calcium entry blockers. Improvement of insulin sensitivity and reduction of plasma insulin levels are desirable attributes of antihypertensive therapy that should be more carefully considered in the future.


Comparison of hemagglutinating pili of Haemophilus influenzae type b with similar structures of nontypeable H. influenzae.

Thirty-eight clinical isolates of nontypeable Haemophilus influenzae were tested for the presence of hemagglutinating pili similar to those of H. influenzae type b (Hib) that mediate buccal epithelial cell adherence. Four endogenously hemagglutinating (HA+) strains were identified, and eight additional HA+ variants were obtained from HA- strains by erythrocyte enrichment. All 12 HA+ nontypeable H. influenzae isolates bound antisera directed against denatured pilins of Hib, but none bound antisera against assembled native pili of Hib. In erythrocyte- and buccal-cell-binding assays, HA+ nontypeable H. influenzae binding was reduced compared with HA+ Hib binding and was not significantly different from HA- nontypeable H. influenzae binding. Both HA- and HA+ nontypeable H. influenzae binding was increased over binding of HA- Hib. HA+ nontypeable H. influenzae strains agglutinated adult erythrocytes that possess the Anton antigen, which is thought to be the receptor for Hib pili, and did not agglutinate cord or Lu(a-b-) dominant erythrocytes, which lack the Anton antigen. Electron microscopy of HA- and HA+ variants of three nontypeable H. influenzae strains showed few or no surface appendages on the HA- organisms, but piluslike structures were seen on many organisms from two HA+ nontypeable H. influenzae strains and on a few organisms from one strain. Thus, nontypeable H. influenzae appears to possess structures that are immunologically similar to the pilins that make up the hemagglutinating pili of Hib. However, nontypeable H. influenzae appears to also possess mechanisms for erythrocyte and buccal cell adherence that are not directly correlated with the presence of a hemagglutinating pilus.


Virulence of non-type 1-fimbriated and nonfimbriated nonflagellated Salmonella typhimurium mutants in murine typhoid fever.

The virulence of Salmonella typhimurium mutants that were unable to synthesize type 1 fimbriae was tested in a murine typhoid fever model. Nonfimbriated mutants (fim) exhibited a lower 50% lethal dose than a wild-type (fim+) strain and produced significantly higher mortality (fim, 55%; fim+, 37% [P less than 0.002]) in mice that were challenged orally. There was no difference in virulence when the wild-type and mutant strains were injected intraperitoneally into mice. The progress of a short-term lethal infection was monitored after oral inoculation of mice with a mixture containing equivalent numbers of fim+ wild-type and fim mutant bacteria. The results indicated that while both strains colonized the intestinal tract equally well and invaded internal organs, the S. typhimurium fim mutant proliferated in the blood of the mice faster than the fim+ strain. The results of the mixed oral challenge suggested that bacteremia caused by fim+ S. typhimurium was reduced or delayed by the sequestration of the fimbriated bacteria in the spleen, liver, and kidneys. Thus, type 1 fimbriae were not virulence factors for S. typhimurium in this model, and the fimbriae may be an impediment to the pathogen in this setting. An S. typhimurium double mutant lacking type 1 fimbriae and flagella (fla) also was tested in mice. The virulence of the fim fla mutant was greatly reduced compared with that of the wild-type strain (mortality from fim fla challenge, 11% [P less than 0.0005]). The significance of this latter result is discussed in relation to host adaptation by pathogenic salmonellae.


Avian P1 antigens inhibit agglutination mediated by P fimbriae of uropathogenic Escherichia coli.

Whole egg white from pigeon, dove, and cockatiel eggs, as well as the ovomucoid fraction of pigeon egg white, exhibited strong P1 antigenic activities and inhibited agglutination of human P1 erythrocytes and of digalactoside-coated latex beads by P-fimbriated Escherichia coli strains. In contrast, chicken egg white exhibited only weak P1 antigenic activity and had little impact on P-fimbrial agglutination. These preparations did not affect hemagglutination by E. coli strains expressing mannose-resistant adhesins other than P fimbriae, i.e., Dr, F1845, and S adhesins. Human anti-P1 serum diminished the P-fimbrial inhibitory activities of pigeon egg white and pigeon ovomucoid. Pigeon ovomucoid was equipotent on a molar basis with globoside, and the pigeon, dove, and cockatiel egg white preparations were equipotent with each other in P-fimbrial inhibition. Incubation of p erythrocytes in whole egg whites or in pigeon ovomucoid did not render them agglutinable by P-fimbriated bacteria, whereas incubation in globoside did. These data demonstrate that whole egg whites (and their ovomucoid fraction) from members of the families Columbidae (pigeons and doves) and Psittacidae (parrots) specifically and potently inhibit P-fimbrial agglutination, probably by providing P1 antigen as a receptor for the P-fimbrial adhesin. Avian egg white preparations may facilitate adhesin characterization of wild-type uropathogenic strains and may useful in preventing upper urinary tract infections due to P-fimbriated E. coli.


Effects of cholinergic and adrenergic agonists on adenylate cyclase activity of retinal microvascular pericytes in culture.

Pericytes are contractile cells that might help regulate microvascular blood flow. To understand their potential role in the regulatory responses of the retina and optic nerve head vessels, the response of pericytes isolated from bovine retinal microvessels was determined to oxotremorine, isoproterenol, phenylephrine, and clonidine. Isoproterenol doubled the basal levels of cyclic adenosine monophosphate (cAMP) specifically through beta-adrenergic receptors, because the effect was blocked by dl-propranolol. The alpha 1 agonist phenylephrine did not induce any major change in adenylate cyclase activity. The alpha 2 agonist clonidine decreased basal cAMP synthesis and reduced the effect of isoproterenol. The cholinergic agonist oxotremorine did not modify the basal activity of adenylate cyclase but was able to decrease by almost 50% the forskolin-induced increase of cAMP. These results suggest that pericytes have functional adrenergic and cholinergic receptors, and they might respond to autonomic vasoactive substances present in vivo.


Agonist response of human isolated posterior ciliary artery.

The isometric responses of isolated human posterior ciliary artery to adrenergic agonists, histamine (HIS), and 5-hydroxytryptamine (5-HT) were studied in passively stretched ring segments mounted in a myograph bath. Cumulative dose response curves were measured for nine agonists: HIS, 5-HT, dopamine (DOPA), epinephrine (A), norepinephrine (NA), tyramine (TYR), phenylephrine (PHE), isoproterenol (ISOP), and xylazine (XYL), and the log(molar concentration) at which one half of the maximum active tension was developed (EC50) was estimated. The ring segments were unresponsive to DOPA and XYL; HIS and ISOP produced biphasic responses with a mild relaxation for low concentrations and small contractions for high concentrations of the agonist. The remaining agonists caused contractile responses of magnitude listed in the rank order following compared with the maximum active tension in response to 0.124 M K(+)-Krebs: Kmax much greater than A greater than 5-HT = PHE greater than NA greater than TYR It was concluded that functional HIS, alpha 1-adrenergic, and 5-HT receptors were present on human posterior ciliary artery but that there are no alpha 2-adrenergic receptors.


A relationship between asparagine synthetase A and aspartyl tRNA synthetase.

A highly conserved protein motif characteristic of Class II aminoacyl tRNA synthetases was found to align with a region of Escherichia coli asparagine synthetase A. The alignment was most striking for aspartyl tRNA synthetase, an enzyme with catalytic similarities to asparagine synthetase. To test whether this sequence reflects a conserved function, site-directed mutagenesis was used to replace the codon for Arg298 of asparagine synthetase A, which aligns with an invariant arginine in the Class II aminoacyl tRNA synthetases. The resulting genes were expressed in E. coli, and the gene products were assayed for asparagine synthetase activity in vitro. Every substitution of Arg298, even to a lysine, resulted in a loss of asparagine synthetase activity. Directed random mutagenesis was then used to create a variety of codon changes which resulted in amino acid substitutions within the conserved motif surrounding Arg298. Of the 15 mutant enzymes with amino acid substitutions yielding soluble enzyme, 13 with changes within the conserved region were found to have lost activity. These results are consistent with the possibility that asparagine synthetase A, one of the two unrelated asparagine synthetases in E. coli, evolved from an ancestral aminoacyl tRNA synthetase.


Stimulation of protein synthesis in COS cells transfected with variants of the alpha-subunit of initiation factor eIF-2.

The role of eukaryotic initiation factor 2 (eIF-2) phosphorylation in translational control has been demonstrated in vivo by overexpressing variant forms of eIF-2 alpha that are not phosphorylated. COS-1 cells transiently transfected with expression vectors for human eIF-2 alpha contain 10-20-fold more eIF-2 alpha subunit than the endogenous COS cell eIF-2 trimeric complex. Expression of the variant form of eIF-2 alpha, Ser51Asp, where Asp replaces Ser51, causes inhibition of protein synthesis, whereas the Ser48Asp variant does not. When either Ser48 or Ser51 is replaced by Ala, the variants stimulate dihydrofolate reductase synthesis when the eIF-2 alpha kinase, DAI, is activated. In order to elucidate these mechanisms, we have separated eIF-2 trimeric complexes from free overexpressed eIF-2 alpha subunits by fast protein liquid chromatography Superose chromatography. Pulse-labeled cells transfected with wild-type or variant DNAs produced eIF-2 preparations with greater than 10-fold higher specific radioactivity in the alpha-subunit compared to the gamma-subunit, thus demonstrating that the human eIF-2 alpha produced from the plasmids readily exchanges into COS cell eIF-2 complexes. Both wild-type and Ser48Ala variant forms of the free 2 alpha-subunit, further purified by MonoQ chromatography, are poor substrates for the heme-regulated eIF-2 alpha kinase, HRI, but are good substrates for double-stranded RNA-activated inhibitor in vitro; the Ser51Ala variant subunit is not phosphorylated by either kinase. None of the purified free eIF-2 alpha subunits inhibits phosphorylation of eIF-2 in vitro, even at up to 8-fold molar excess. Examination of the extent of eIF-2 alpha phosphorylation in the COS cell eIF-2 complexes by two-dimensional polyacrylamide gel electrophoresis shows that the stimulation of dihydrofolate reductase synthesis by the Ser51Ala variant is most readily explained by failure of eIF-2 to be phosphorylated. Stimulation by the Ser48Ala variant appears to occur by mitigation of the effect of phosphorylation at Ser51 since the double variant, Ser48Ala-Ser51Asp, inhibits protein synthesis less than the single variant Ser51Asp. The evidence argues strongly against there being a second site of phosphorylation involved in translational repression.


Heat sensitivity and Sp1 activation of complex formation at the Syrian hamster carbamoyl-phosphate synthase (glutamine-hydrolyzing)/aspartate carbamoyltransferase/dihydroorotase promoter in vitro.

To study the regulation of transcription of the carbamoyl-phosphate synthase (glutamine-hydrolyzing)/aspartate carbamoyltransferase/dihydroorotase (CAD) gene from the Syrian hamster, Mesocricetus auratus, we developed a homologous in vitro transcription system on the basis of nuclear extract from Syrian hamster kidney cells. We optimized the reaction temperature and the concentrations of DNA template, KCl, and MgCl2 simultaneously with the response surface method and found an unusually low temperature optimum of 20 degrees C. We therefore investigated whether CAD transcription in vitro depended on a heat-labile component of nuclear extract. Preincubating extract alone at 30 degrees C reduced transcription from the CAD promoter but not from the major late promoter of adenovirus 2. The formation of stable initiation complexes at the CAD promoter was diminished in heat-treated extract; run-off transcripts, however, accumulated at the same rate as in untreated extract. The heat sensitivity of complex formation correlated with the heat sensitivity of DNA binding by transcription factor Sp1, which binds to two sites in the CAD promoter; moreover, both preformed initiation complexes and DNA-bound Sp1 were heat-resistant. Adding purified Sp1 to heat-treated extract restored complex formation. We propose that Sp1 activates CAD transcription by stabilizing initiation complexes at the CAD promoter.


Mammalian mitochondrial chaperonin 60 functions as a single toroidal ring.

Chaperonins are thought to participate in the process of protein folding in bacteria and in eukaryotic mitochondria and chloroplasts. While some chaperonins are relatively well characterized, the structures of the mammalian chaperonins are unknown. We have expressed a mammalian mitochondrial chaperonin 60 in Escherichia coli and purified the recombinant protein to homogeneity. Structural and biochemical analyses of this protein establish a single toroidal structure of seven subunits, in contrast to the homologous bacterial, fungal, and plant chaperonin 60s, which have double toroidal structures comprising two layers of seven identical subjects each. The recombinant mammalian chaperonin 60, together with the mammalian chaperonin 10 (but not with bacterial chaperonin 10), facilitates the formation of catalytically active ribulose-bisphosphate carboxylase from an unfolded state in the presence of K+ and MgATP. Analysis of the partial reactions involved in this in vitro reconstitution reveals that the single toroid of chaperonin 60 can form stable complexes with both unfolded or partially folded [35S]ribulose-bisphosphate carboxylase and mitochondrial (but not bacterial) chaperonin 10 in the presence of MgATP. We conclude that the minimal functional unit of chaperonin 60 is a single hepatmeric toroid.


An initiation codon mutation in CD18 in association with the moderate phenotype of leukocyte adhesion deficiency.

Leukocyte adhesion deficiency (LAD) is an autosomal recessive disease caused by mutations in the CD18 gene which codes for the beta 2 integrin subunit. We studied two patients, the first of which had a moderate LAD phenotype and expressed only 9% of CD11/CD18 on blood leukocytes. RNA from lymphoblasts was reverse-transcribed, and the cDNA was amplified, cloned, and sequenced. An ATG to AAG alteration in the initiation codon was detected in 39 of 45 (87%) cDNA clones. This mutation was detected in the father, but not in the mother. The maternal defect was shown to be a frameshift mutation with the deletion of a single T in the aspartic acid codon at position 690 (GAT), 11 amino acids N-terminal to the beginning of the transmembrane domain. This mutation predicts a polypeptide which would terminate without transmembrane or cytoplasmic domains. The frameshift mutation was also found in the second patient who had the severe phenotype of LAD (less than 1% of CD11/CD18), indicating that this allele does not encode a functional protein. The partial expression in the patient with a moderate phenotype must be derived from the initiation codon mutation and may be due to a low level of initiation of translation of the CD18 mRNA at the second codon (CUG).


A single histidine in GABAA receptors is essential for benzodiazepine agonist binding.

Benzodiazepines (BZ) modulate neurotransmitter-evoked chloride currents at the gamma-aminobutyric acid type A (GABAA) receptor, the major inhibitory ion channel in the mammalian brain. This receptor is composed of structurally distinct subunits whose numerous molecular variants underlie the observed diversity in the properties of the BZ site. Pharmacologically distinct BZ sites can be recreated by the recombinant coexpression of any one of six alpha subunits, a beta subunit variant, and the gamma 2 subunit. In these receptors the alpha variant determines the affinity for ligand binding of the BZ site. Notably, the alpha 1 and alpha 6 variants impart on alpha chi beta 2 gamma 2 receptors high and negligible affinity, respectively, to BZ ligands with sedative as well as anxiolytic activities. By exchanging domains between the alpha 1 and alpha 6 variants, we show that a portion of the large extracellular domain determines sensitivity toward these ligands. Furthermore, we identify a single histidine residue in the alpha 1 variant, replaced by an arginine in alpha 6, as a major determinant for high affinity binding of BZ agonists. This residue also plays a role in determining high affinity binding for BZ antagonists. Hence, this histidine present in the alpha 1, alpha 2, alpha 3, and alpha 5 subunits appears to be a key residue for the action of clinically used BZ ligands.


Constitutive activation of the alpha 1B-adrenergic receptor by all amino acid substitutions at a single site. Evidence for a region which constrains receptor activation.

Mutations in an intracellular region of the alpha 1B-adrenergic receptor constitutively activate the receptor, resulting in G protein coupling in the absence of agonist, as evidenced by elevated levels of polyphosphoinositide hydrolysis. Remarkably, all 19 possible amino acid substitutions at a single site in this region (alanine 293) confer constitutive activity. This set of mutated receptors exhibits a graded range of elevated biological activities, apparently representing a spectrum of receptor conformations which mimic the "active" state of the wild type receptor. In addition to their constitutive activities, these mutated receptors all demonstrate a higher affinity for agonists, another primary characteristic of the "active" conformation of G protein-coupled receptors. The fact that all possible mutations at this particular site result in increased activity suggests that this region may function to constrain the G protein coupling of the receptor, a constraint which is normally relieved by agonist occupancy.


Different positively charged amino acids have similar effects on the topology of a polytopic transmembrane protein in Escherichia coli.

Integral membrane proteins from a wide variety of sources conform to a "positive-inside rule," with many more positively charged amino acids in their cytoplasmic as compared to extracytoplasmic domains. A growing body of experimental work also points to positively charged residues in regions flanking the apolar transmembrane segments as being the main topological determinants. In this paper, we report a systematic comparison of the effects of positively (Arg, Lys, His) as well as negatively (Asp, Glu) charged residues on the membrane topology of a model Escherichia coli inner membrane protein. Our results show that positive charge is indeed the major factor determining the transmembrane topology, with Arg and Lys being of nearly equal efficiency. His, although normally a very weak topological determinant, can be potentiated by a lowering of the cytoplasmic pH. Asp and Glu affect the topology to similar extents and only when present in very high numbers.


pH-dependent heterogeneity of acidic amino acid transport in rabbit jejunal brush border membrane vesicles.

Initial rates of Na(+)-dependent L-glutamic and D-aspartic acid uptake were determined at various substrate concentrations using a fast sampling, rapid filtration apparatus, and the resulting data were analyzed by nonlinear computer fitting to various transport models. At pH 6.0, L-glutamic acid transport was best accounted for by the presence of both high (Km = 61 microM) and low (Km = 7.0 mM) affinity pathways, whereas D-aspartic acid transport was restricted to a single high affinity route (Km = 80 microM). Excess D-aspartic acid and L-phenylalanine served to isolate L-glutamic acid flux through the remaining low and high affinity systems, respectively. Inhibition studies of other amino acids and analogs allowed us to identify the high affinity pathway as the X-AG system and the low affinity one as the intestinal NBB system. The pH dependences of the high and low affinity pathways of L-glutamic acid transport also allowed us to establish some relationship between the NBB and the more classical ASC system. Finally, these studies also revealed a heterotropic activation of the intestinal X-AG transport system by all neutral amino acids but glycine through an apparent activation of Vmax.


Oxidative modification of Escherichia coli glutamine synthetase. Decreases in the thermodynamic stability of protein structure and specific changes in the active site conformation.

Metal catalyzed oxidation of specific amino acid residues has been proposed to be an important physiological mechanism of marking proteins for proteolytic degradation. After initial oxidative inactivation of dodecameric Escherichia coli glutamine synthetase (GS), the integrity of the GS active site and protein structure was assessed by monitoring ATP binding, observing a susceptibility of GS to tryptic cleavage, and comparative thermodynamic analysis. The tryptic cleavage rates of an active site linked central loop were significantly accelerated for the oxidized conformer. This tryptic cleavage was essentially prevented in the presence of glutamate for native GS but not for the oxidized conformer. The integrity of the ATP binding site in the oxidized GS was substantially altered as indicated by the reduction in fluorescence enhancement associated with ATP binding. Decreases in the free energies of quaternary protein structure and subunit interactions due to oxidative modification were determined by temperature and urea induced unfolding equilibrium measurements. Comparative thermal stability measurements of a partial unfolding transition indicated that the loss in stabilization free energy for the oxidized GS conformer was 1.3 kcal/mol dodecamer. Under alkaline conditions, the urea-induced disruption of quaternary and tertiary structures of oxidized and native GS were examined. This comparative analysis revealed that the free energies of the subunit interactions and unfolding of the dissociated monomers for oxidized GS were decreased by 1.5 and 1.7 kcal/mol, respectively. Our results suggest that small free energy decreases in GS protein structural stability of only 1-2 kcal/mol may be responsible for the selective proteolytic turnover of the oxidized GS.


Beta-adrenergic, cAMP-mediated stimulation of proliferation of brown fat cells in primary culture. Mediation via beta 1 but not via beta 3 adrenoceptors.

The ability of adrenergic stimulation to affect the rate of DNA synthesis in mouse brown adipocyte precursor cells proliferating in primary culture was investigated. Addition of 1 microM norepinephrine to the cells at day 4 in culture (proliferating cells) significantly increased the rate of DNA synthesis, whereas no significant effect was seen at day 9 (confluent cells). The effect of norepinephrine could be mimicked by forskolin, cholera toxin, and by cAMP analogues. Specific [3H]thymidine incorporation (per unit of DNA) was reduced by norepinephrine stimulation, indicating saturation of the salvage pathway for dTTP synthesis already in unstimulated cells and implying a beta-adrenergic stimulation of dTTP synthesis. Pharmacological characterization of this effect indicated it was mediated by beta 1 receptors, with alpha 2 receptors exerting an opposing effect. Notably, the stimulation of DNA synthesis was not observed with the beta 3-specific agonist CGP-12177. In contrast, both norepinephrine and CGP-12177 were able to induce the expression of the uncoupling protein thermogenin in the confluent cultured cells. This coincided with a shift in the cAMP-elevating potential of CGP-12177, from being antagonistic to norepinephrine stimulation in proliferating cells to being itself a full agonist in confluent cells, implying occurrence of coupled beta 3 receptors as part of the differentiation process. It was concluded that brown fat precursor cells respond directly to norepinephrine stimulation with an increased DNA synthesis, and that this response is mediated via the classical beta 1 receptors. This probably represents the cellular basis for the hyperplasia observed in the tissue in physiologically recruited states.


Divalent cation regulation of the function of the leukocyte integrin LFA-1.

The integrin lymphocyte function-associated antigen-1 (LFA-1) expressed on T cells serves as a useful model for analysis of leukocyte integrin functional activity. We have assessed the role of divalent cations Mg2+, Ca2+, and Mn2+ in LFA-1 binding to ligand intercellular adhesion molecule-1 (ICAM-1) and induction of the divalent cation-dependent epitope recognized by mAb 24. Manganese strongly promoted both expression of the 24 epitope and T cell binding to ICAM-1 via LFA-1, suggesting that Mn2+ is able to directly alter the conformation of LFA-1 in a manner that favors ligand binding. Since Mn2+ also promotes functional activity of other integrins, parallels in mechanism of ligand binding may span the integrin family. In contrast, induction of 24 epitope expression by Mg2+ required removal of Ca2+ from T cell LFA-1 with EGTA. Furthermore, binding of mAb 24 to T cell LFA-1 in the presence of either Mn2+ or Mg2+ was found to be specifically inhibited by Ca2+, suggestive of a negative regulatory role for Ca2+ in the control of leukocyte integrin function. Analysis of T cell binding to ICAM-1 via LFA-1 in the presence of Mg2+ or Mn2+, confirmed that Ca2+ exerted inhibitory effects upon LFA-1 function. The implication of our findings is that Ca2+ bound with relatively high affinity to LFA-1 may serve to maintain an inactive state. Thus induction of function and 24 epitope expression may occur as a result of displacement of Ca2+ from leukocyte integrins or alternatively, such activators may be able to impose the required conformational change in the presence of bound Ca2+.


Density-dependent regulation of cell surface gamma-glutamyl transpeptidase in cultured glial cells.

A decline in cell surface gamma-glutamyl transpeptidase specific activity was previously observed to be concomitant with C6 glial cell proliferation. To elucidate the underlying factor(s) mediating gamma-glutamyl transpeptidase down-regulation, the effects of C6 cell density and culture conditions on cell surface transpeptidase activity levels were investigated. After 24 h of culture, the transpeptidase specific activities were inversely related to the initial plating densities. The lower-density cultures showed an induction within 24 h of plating. As the cultures proliferated, the specific transpeptidase activities declined to a common low level at post-confluency. The gamma-glutamyl transpeptidase down-regulation was unrelated to cell growth rate and was most pronounced during logarithmic proliferation. Induction and down-regulation of gamma-glutamyl transpeptidase activity at low cell densities were not a result of trypsinization. Supplementation of low-density cultures with conditioned medium, use of matrix-coated wells, or periodic replacement of growth media to prevent conditioning had minor effects on the decline of cell surface activity. Kinetic analysis showed that the Michaelis constants and the reaction mechanism were unaltered by cell density, indicating that down-regulation was not due to allosteric factors or an alteration in enzyme character. A reduction in the maximal velocity of cell surface transpeptidation at higher cell densities suggested that gamma-glutamyl transpeptidase down-regulation is related to the concentration of enzyme at the cell surface. Immunocytochemical localization of gamma-glutamyl transpeptidase demonstrated that gamma-glutamyl transpeptidase antigen levels decrease as C6 cell density increases. These results led us to propose that cell-cell contact stimulates the disappearance of gamma-glutamyl transpeptidase from the surface of cultured C6 glial cells.


Loss of sperm in juvenile spermatogonial depletion (jsd) mutant mice is ascribed to a defect of intratubular environment to support germ cell differentiation.

C57BL/6(B6)-jsd/jsd mice are sterile due to the defective spermatogenesis in the testes. To know the cause of the deficient spermatogenesis in B6-jsd/jsd mice, we examined whether the problem is within or outside the seminiferous tubules by transplanting tubules from cryptorchid testes of B6- +/+ mice into B6-jsd/jsd testes or tubules from B6-jsd/jsd mice into testes of (WB x C57BL/6)F1-W/Wv (hereafter, WBB6F1-W/Wv) mice. Type A spermatogonia differentiated into spermatids in seminiferous tubules from cryptorchid testes transplanted into B6-jsd/jsd testes. In contrast, in B6-jsd/jsd tubules transplanted into WBB6F1-W/Wv testes, type A spermatogonia were stimulated to mitotic proliferation, but didn't proceed to any differentiated germ cells. The present results suggest that the cause of the deficient spermatogenesis in B6-jsd/jsd mice is a defect of intratubular environment to support germ cell differentiation.


Gastroesophageal reflux: diagnosis and management.

The pathologic potential of chronic esophagitis cannot be overemphasized. Persistent reflux causing cycles of mucosal damage followed by healing may eventually lead to end-stage disease, with development of peptic stricture. The most effective drugs available are those that inhibit acid production. Drugs that will enhance lower esophageal function are still in the future.


Stimulated growth hormone (GH) secretion in children with delays in pubertal development before and after the onset of puberty: relationship with peripheral plasma GH-releasing hormone and somatostatin levels.

A reduced GH secretion has often been shown in prepubertal children with delays in pubertal development. In order to study the mechanism underlying this finding, we evaluated peripheral circulating levels of GH, GHRH, and somatostatin (SRIH) before and after the onset of sexual development in a group of eight late maturing children (six boys, two girls), comparing the results with those obtained in two groups of five prepubertal and four pubertal short children with familial short stature. GH was measured by a two-site immunoradiometric assay. Both GHRH and SRIH were assayed by specific RIAs after an acetone-petrolether extraction from plasma. Our data showed a significant increase (P less than 0.001) in GH, GHRH, and SRIH levels (peak vs. basal values) in response to L-dopa administration in all groups. In pubertal children with delays in pubertal development GH and GHRH peak values (15.8 +/- 2.2 micrograms/L and 120 +/- 18 pg/mL, respectively) were significantly greater (P less than 0.001) than in the same subjects before puberty (8.2 +/- 0.9 micrograms/L and 79 +/- 9 pg/mL, respectively), whereas SRIH peak values did not significantly change (41 +/- 6 vs. 41 +/- 5 pg/mL; P = NS). Furthermore, prepubertal subjects with delays in pubertal development showed GH and GHRH peak values lower (P less than 0.001) than those of prepubertal subjects with FSS (13.3 +/- 1.8 micrograms/L and 120 +/- 13 pg/mL, respectively), whereas no statistical difference was present between the two groups of subjects after pubertal development (18.2 +/- 2.9 micrograms/L and 128 +/- 11 pg/mL, respectively). In conclusion, these findings support the assumption that in late maturing subjects during prepubertal period the decreased GH secretion may be ascribed to a reduced GHRH secretion, reversible with the onset of puberty, without change in circulating SRIH levels.


Tumor necrosis factor beta gene polymorphisms in Graves' disease.

The physical mapping of tumor necrosis factor alpha (TNF alpha) and lymphotoxin (TNF beta) genes to the short arm of chromosome 6 in man between the loci for histocompatibility leucocyte antigens (HLA)-B and the complement system focused attention to this genetic region that controls immune responses in many ways. It also holds susceptibility genes for a variety of autoimmune disorders that are linked to specific alleles of loci in the HLA D subregion. We have recently identified a TNF restriction fragment length polymorphism with the enzyme NcoI (K. Badenhoop, G. Schwarz, J. Trowsdale, et al. Diabetologia. 1989;32:445-8). The less frequent fragment of 5.5 kilobase (kb) is in strong linkage disequilibrium with the HLA haplotype A1B8DR3. Since Graves' disease is linked to A1B8DR3, we analyzed TNF gene polymorphisms in a large group of Graves' disease patients and normal controls derived from four Centers. We show here a significant association of TNF beta polymorphisms with Graves' disease. The patients have less homozygotes for the 10.5 kb band (60 of 174, 34%) and more heterozygotes 10.5/5.5 kb (96 of 174, 55%), than 173 controls (49% homozygotes 10.5 kb and 42% heterozygotes; chi 2 = 7.45, P less than 0.03). When DR3+ patients and controls were analyzed separately, heterozygotes were still significantly increased in DR3+ Graves' disease patients (54 of 77, 70%) compared to DR3+ controls (21 of 45, 47%; chi 2 = 6.6, P less than 0.04). Furthermore, TNF fragment heterozygotes were found predominantly in patients, who had TSH-receptor antibodies (29/45, 64%, P less than 0.007), implying that these patients might represent an immunogenetic subset of the disease. Although TNF beta polymorphisms are linked to A1B8DR3, these results suggest that they represent an additional susceptibility marker in Graves' disease.


Presymptomatic testing using DNA markers for individuals at risk for familial multiple endocrine neoplasia 2A.

The carrier status of 39 at-risk individuals in 6 multiple endocrine neoplasia 2A families was determined using a DNA based test. We were able to calculate a virtual diagnosis (probability greater than 95%) for 77% of the individuals and a probable diagnosis (probability greater than 90%) for 90% of the individuals. This study points out some of the problems of specific pedigree structures that can affect the risk calculation. This study further shows that no single test based on either biochemistry, pathology, or genetics can consistently and unambiguously produce a presymptomatic diagnosis. We also describe two specific examples where DNA testing has helped to resolve clinical uncertainties in at-risk individuals.


Differential changes in alpha- and beta-adrenoceptor linked [45Ca2+] uptake in platelets from patients with anorexia nervosa.

[45Ca2+] Uptake was studied in response to adrenaline, isoprenaline, noradrenaline, and (Bu)2cAMP in platelets from patients with anorexia nervosa. In both controls and anorectics, adrenaline, isoprenaline, noradrenaline, and (Bu)2cAMP stimulated [45Ca2+] uptake. In receptor subtype characterisation studies on control platelets, adrenaline-stimulated [45Ca2+] uptake was blocked by yohimbine (an alpha 2-adrenoceptor antagonist) and the specific beta 2-adrenoceptor antagonist ICI 118,551, but not by atenolol (a beta 1-antagonist). Isoprenaline action was blocked by ICI 118,551, but not by yohimbine. Noradrenaline-stimulated [45Ca2+] uptake was blocked by yohimbine but not by ICI 118,551. In platelets from anorectic patients, there was a significant increase in noradrenaline-stimulated [45Ca2+] uptake, a significant diminution in adrenaline and isoprenaline-stimulated [45Ca2+] uptake, but no significant difference in (Bu)2cAMP-stimulated [45Ca2+] uptake, when compared with controls. Basal uptake was also significantly enhanced in anorectics and was found to be inhibited with verapamil but not adrenoceptor antagonist. These data firstly indicate that both alpha 2- and beta 2-adrenoceptor activation elicits [45Ca2+] uptake by platelets. It is proposed that this stimulated [45Ca2+] uptake does not reflect changes in cytosolic Ca2+ but to localized changes of Ca2+ at the plasma membrane, possibly associated with receptor activation, per se. The respective increase and decrease of alpha- and beta-adrenoceptor activity in platelets from anorectic patients is in accord with other reports of changes of adrenoceptor number and type in platelets and other cells from anorectic patients. There may also be an increase in calcium channel activity in platelets from anorectics.


Elevation of IgE in HIV-infected subjects: a marker of poor prognosis.

The IgE synthesis is tightly controlled by a complex network of T and B cells. Because human immunodeficiency virus (HIV) disease associates T cell activation and depletion, polyclonal B cell activation, atopic symptoms, drug hypersensitivity, and autoimmune activity, we have evaluated IgE, as well as IgA, IgG, and IgM, in 315 HIV-seropositive individuals with or without acquired immunodeficiency syndrome (AIDS) and compared the results to those of 100 HIV-seronegative subjects. IgE levels were higher in HIV-infected subjects as a whole, compared to levels in seronegative control subjects (p less than 0.05). This difference was particularly marked between patients with AIDS and control subjects (p less than 0.005). A strong relationship appeared between IgE and the immune status as assessed by CD4 cell counts (p less than 0.001 between IgE values in patients with CD4 less than 300 or greater than 300/microliters). In addition, we assessed the predictive value of IgE elevation over disease progression: in subjects with a CD4 count less than 300/microliters, the survival analysis disclosed a 24-month occurrence rate of AIDS of 83% in individuals with IgE greater than 150 KIU/L versus 44% in individuals with IgE less than 150 (p = 0.016). In subjects with an AIDS-related complex, IgE greater than 150 indicated a 100% rate of AIDS versus 9% in individuals with IgE less than 150 (p = 0.003). Thus, IgE levels appear to be a very discriminative marker between patients in late stages of HIV infection.(ABSTRACT TRUNCATED AT 250 WORDS)


Metabolic half-life of somatostatin and peptidase activities are altered in Alzheimer's disease.

Several reports have described decreased immunoreactive somatostatin levels in specific regions of post-mortem brain tissue from patients diagnosed with senile dementia of the Alzheimer type (SDAT). In an attempt to determine if the metabolism of somatostatin is also altered as a result of SDAT, we examined the regional metabolic half-life of somatostatin-28 (SS-28) and somatostatin-14 (SS-14). The activity of the following peptidases was also determined: neutral endopeptidase E.C. 3.4.24.11; metalloendopeptidase E.C. 3.4.24.15; carboxypeptidase E (E.C. 3.4.17.10); and trypsin-like serine protease. The metabolic half-life of SS-28 was significantly reduced in post-mortem Brodmann Area 22 of SDAT tissue. This decrease in SS-28 metabolic half-life was correlated with a significant increase in trypsin-like serine protease activity in the same SDAT brain region. The formation rate of SS-14 from SS-28 incubated with Brodmann Area 22 homogenates was also increased in SDAT tissues as compared to controls. A regional variation in neutral endopeptidase E.C. 3.4.24.11 was also noted in both controls and SDAT samples. Although postmortem intervals of samples varied significantly, no effect was seen on any biochemical parameter measured. Results from this study provide evidence that a correlation can be made between changes in metabolic half-life somatostatin and alterations in neuropeptidase activities due to SDAT. As these data show alterations in both proteolytic metabolism and peptidase activities, many other biologically active peptide substrates could also be affected in SDAT.