Human T-cell lymphotropic virus (HTLV)-related endogenous sequence, HRES-1, encodes a 28-kDa protein: a possible autoantigen for HTLV-I gag-reactive autoantibodies.

The presence of a human T-cell lymphotropic virus (HTLV)-related endogenous sequence, HRES-1, in the human genome has been documented. The HRES-1 genomic locus is transcriptionally active and contains open reading frames. Antibodies 232 and 233, specific for synthetic peptides pep14-24 and pep117-127, corresponding to two nonoverlapping HTLV-related regions in the longer open reading frame of HRES-1, recognize an identical 28-kDa protein in H9 human T cells. Thus, HRES-1 is a human endogenous retroviral sequence capable of protein expression. HRES-1/p28 is localized to the cytoplasm and nuclear bodies. While HTLV-I-specific antibodies react with HRES-1 peptides, antibody 233 cross-reacts with HTLV-I gag p24 protein. Three consecutive highly charged amino acid residues, Arg-Arg-Glu, present in both HRES-1 pep117-127 and HTLV-I gag p24 are likely to be the core of cross-reactive epitopes. The prevalence of antibodies to HRES-1 peptides pep14-24 and pep117-127 was determined in 65 normal blood donors and 146 patients with immunological disorders. Sera of patients with multiple sclerosis (19 out of 65, 29%), progressive systemic sclerosis (4 out of 17, 23%), systemic lupus erythematosus (4 out of 19, 21%), and Sjogren syndrome (2 out of 19, 10%) contained significantly higher HRES-1 peptide binding activity than sera of normal donors. Sera of patients with AIDS showed no specific binding to HRES-1 peptides. Nine of 30 HRES-1-seropositive patients showed immunoreactivity to HTLV-I gag p24. The data indicate that HRES-1/p28 may serve as an autoantigen eliciting autoantibodies cross-reactive with HTLV-I gag antigens.


Analysis of HLA class II allogenotyping in Australian aborigines and Papua New Guinea populations.

Human leukocyte antigen (HLA) class II allogenotyping has been applied to investigate the polymorphism of the DRB, DQB1, DQB2, DQA1, and DQA2 genes in Aborigines from the East Coast of Australia and in Melanesians from the Papua New Guinea North-East Coast and Highlands. Three new DR/DQ arrangements were observed, DRw14/DQB1-2b/DQA1-1a and DRw5Nauru/DQB1-3a/DQA1-2 (n Australian Aborigines), and DRw5Nauru/DQB1-1a/DQA1-1b (in Madang). DQA2 and DQB2 allogenotyping with TaqI and PstI digested genomic DNA revealed little polymorphism among the Papua New Guineans, with DQA2-Xa1 and DQB2-Xb1 the most common alleles in all the groups. However, the presence of DQA2-Xa2 in Papuans and Australian Aborigines reflects the degree of admixture with Caucasoids while the DQA2-Xa4 allele in Madang is probably a marker of Mongoloid origin.


Questions about inhaled beta 2-adrenoceptor agonists in asthma.

The safety of the most widely prescribed antiasthma drugs, beta 2-adrenoceptor agonists, has recently been questioned. Issues such as their suitability for long-term and regular prophylactic use are addressed in this Comment article by Peter Barnes and Fan Chung, who examine the possibility that the beta 2-agonists themselves contribute to worsening symptoms in asthma patients, thus setting up a vicious circle with greater use of the drugs. They conclude that it would be prudent to restrict the use of beta 2-agonists in asthma to on-demand immediate symptom control.


Distemper encephalitis in pups after vaccination of the dam.

A five-year-old labrador bitch which had whelped 10 pups three days previously was given booster vaccination against distemper, adenovirus, parvovirus, parainfluenzavirus and leptospirosis. Eighteen days later, signs of central nervous system disease developed in some of the pups, five of which were ultimately euthanased. The cause of the nervous disease was found to be canine distemper, and serological studies showed that the infection was limited to some members of the litter, suggesting that the vaccinal rather than a field virus was more likely to have been responsible.


Nonsedating antihistamines: pharmacology, clinical efficacy and adverse effects.

Antihistamines are effective therapy for histamine-mediated conditions, including seasonal and perennial allergic rhinitis and chronic urticaria. Until recently, all antihistamines produced some degree of drowsiness, as well as anticholinergic side effects. Several nonsedating antihistamines have been developed. Two of these antihistamines--terfenadine and astemizole--are commercially available. The lack of central nervous system effects is attributed to the inability of these drugs to penetrate the blood-brain barrier. They also have no appreciable binding to cholinergic receptors. Clinical trials have demonstrated that the newer agents are as effective as classic antihistamines and that they have no greater incidence of central nervous system or anticholinergic side effects than placebo.


Brain damage from pertussis immunization. A Canadian neurologist's perspective.

Uncontrolled case series noting an association between diphtheria, tetanus, and pertussis vaccine (DTP) immunization and brain damage have lead to widespread reduction in immunization, epidemics of pertussis and litigation. A judge, hearing the only Canadian court case, concluded there is no evidence that DTP induced brain injury. It is difficult to develop a cogent biologic hypothesis for the mechanism of DTP-induced brain injury, especially when the postulated clinical syndrome is variable without a characteristic neuropathologic pattern. Timed observational, case-controlled, and prospective cohort studies have not shown evidence of brain damage from pertussis vaccine. This suggests that the cause(s) of brain injury recorded in case series was other than DTP immunization. Any relationship is likely a temporal coincidence because DTP is administered several times in infancy. A vigorous effort is required to dispel the myth of DTP-induced brain damage.


Relationship between dideoxyinosine exposure, CD4 counts, and p24 antigen levels in human immunodeficiency virus infection. A phase I trial.

To determine the relation between exposure to dideoxyinosine (ddl) and increased CD4 cell counts and suppression of serum p24 antigen in patients infected with the human immunodeficiency virus (HIV). Open-label, phase I study. Two university hospitals. Patients were studied in both inpatient and outpatient settings. Of 36 HIV-infected patients enrolled, 18 had adequate pharmacokinetic information for analysis. Dideoxyinosine was administered intravenously every 12 hours for 2 weeks. Patients were switched to oral administration at twice the intravenous dose. Pharmacokinetic profiles were obtained twice during each period. A 40-fold range of dose was examined. CD4-positive T-lymphocyte counts and serum p24 antigen levels were determined. Plasma area under the ddl concentration-time curve was determined for a single dose and at steady state. Increases in CD4-positive T-lymphocyte counts were independent of ddl exposure and were proportional to the starting CD4 count. Suppression of circulating p24 antigen was influenced by cumulative exposure to ddl and was statistically significant. The CD4-positive T-lymphocyte count increased at low ddl concentrations or exposures; the extent of this increase was directly proportional to the patient's CD4 count at the start of therapy. Suppression of p24 antigen was related to cumulative exposure to ddl. Therapeutic responses can probably be obtained with ddl, while minimizing long-term toxicity, using daily doses of 10 mg/kg body weight, or less.


The angiotensin AT2 receptor stimulates protein tyrosine phosphatase activity and mediates inhibition of particulate guanylate cyclase.

The signalling mechanism and cellular targets of the AT2 receptor are still unknown. We report that angiotensin II (Ang II) inhibits basal and atrial natriuretic peptide stimulated particulate guanylate cyclase (pGC) activity through AT2 receptors in rat adrenal glomerulosa and PC12W cells. This inhibition is blocked by the phosphotyrosine phosphatase (PTPase) inhibitor orthovanadate but not by the Ser/Thr phosphatase inhibitor okadaic acid, suggesting the involvement of a PTPase in this process. Moreover, Ang II induces a rapid, transient and orthovanadate sensitive dephosphorylation of phosphotyrosine containing proteins in PC12W cells. Our findings suggest that AT2 receptors signal through stimulation of a PTPase and that this mechanism is implicated in the regulation of pGC activity. This observation is also the first example of hormonal inhibition of basal pGC activity.


Studies on cytosolic guanylate cyclase from human placenta.

We have purified the soluble form of guanylate cyclase from human placenta greater than 2400-fold. The enzyme shared several characteristics with the enzyme purified from other sources including molecular mass and subunit composition, activation by divalent cations, inhibition by ATP and Michaelis constants. The enzyme, however, had a lower absorption maximum in the Soret region (417 +/- 1 nm) than the enzyme from other sources and was activated only one-fifth as much by nitric oxide as the bovine lung enzyme. It appears that the heme prosthetic group in the human placental enzyme may be hexa-coordinate and in the bovine lung enzyme the heme group may be penta-coordinate.


[Life saving glance diagnosis in type IIb multiple endocrine neoplasia].

Medullary carcinoma of the thyroid gland is a feature of multiple endocrine neoplasia, type IIb (MEN IIb). The cancer frequently gives rise to metastases in early life. Marfanoid habitus and virtually pathognomic mucosal ganglioneuromas, often situated on the tongue, enable early diagnosis. These stigmata should alert the clinician to the possibility of MEN IIb before medullary carcinoma is clinically manifest. We now believe that it is reasonable to perform a total thyroidectomy in children with the typical physical appearance of this syndrome regardless of age since medullary carcinoma of the thyroid gland appears in almost every case. Calcitonin, a hormone secreted by the C-cells, serves as a plasma tumor marker. Intravenously administered, pentagastrin is a potent secretagogue which is very useful in the early diagnosis of either primary or recurrent medullary carcinoma. With this pentagastrin test, a laboratory screening program is possible allowing the clinician, specialist, to recognize the syndrome.


The same gamma-glutamyl transpeptidase RNA species is expressed in fetal liver, hepatic carcinomas, and rasT24-transformed rat liver epithelial cells.

In rats, gamma-glutamyl transpeptidase (gamma GT) exists as a single-copy gene, and three distinct species of RNA (types I, II, and III) that differ in their 5' untranslated regions have been identified. To compare steady-state levels of these gamma GT RNAs in rat tissues, hepatic carcinomas, and cultured cells, we used RNA dot-blot hybridization and a reverse transcriptase-polymerase chain reaction (RT-PCR) technique with oligonucleotides specifically designed for each type of RNA. Fetal liver, hepatic carcinomas, rasT24-transformed rat liver epithelial (RLE) cells and pancreas make only type III RNA. Liver and untransformed RLE cells do not make detectable levels of gamma GT RNA. We found that both fetal and adult kidneys synthesize all three types of RNA, indicating that increases in gamma GT RNA known to occur after birth do not result from recruitment of additional RNA species. When we increased the sensitivity of the assay approximately 1000 fold by sequencing the RT-PCR product directly after an additional round of amplification, we found that very low levels of types I and II RNA were present in fetal liver, rasT24-transformed RLE cells, and pancreas, and that adult liver and untransformed RLE cells synthesized very low levels of all three RNA species. Rat-1 fibroblasts did not make levels of gamma GT RNA detectable by this method. These results demonstrate that different gamma GT RNA species are regulated differently during development and neoplastic transformation and that there is a commitment in some cell types to very-low-level expression of gamma GT RNAs.


Non-adrenergic, non-cholinergic inhibitory responses to nerve stimulation in rat colonic circular muscle.

The nerve-mediated response to electrical field stimulation (EFS) in rat colonic circular muscle was investigated using the single sucrose-gap technique. EFS with a single pulse (0.4 ms, supramaximal voltage) elicited transient TTX-sensitive hyperpolarization (IJP) often followed by an 'off' depolarization associated with muscular contraction. No relaxation associated with the IJP could be seen unless tone was pharmacologically induced by carbachol (10(-6) M). IJPs were due to non-adrenergic, non-cholinergic (NANC) nerve activation since they were not affected by atropine (10(-7) M) or guanethidine (10(-6) M) superfusion. The mechanism underlying the IJP was presumably an increase in K+ conductance, and the NANC neurotransmitter might open largely apamin-sensitive, Ca(2+)-dependent K+ channels. Purines or vasoactive intestinal polypeptide (VIP) did not mimic the effects of NANC nerve stimulation. Therefore, the NANC inhibitory system, producing IJPs, in rat colonic circular muscle is not purinergic or VIPergic in nature.


Somatostatin and growth hormone regulation in cancer.

Somatostatin analogues are used clinically in a variety of pituitary and gastroenteropancreatic tumours. In addition, they may influence breast and prostate growth either directly through somatostatin receptors or indirectly through inhibition of growth hormone and prolactin release. Somatostatin analogues may interfere with EGF/TGF alpha-stimulated growth of these tumours and can suppress circulating levels of IGF-I in addition.


Classification of vasculitis.

Usually classifications of vasculitic syndromes are based on clinical and histopathologic findings because pathogenetic mechanisms are poorly understood. A subcommittee of the Diagnostic and Therapeutic Criteria Committee of the American College of Rheumatology recently developed classification criteria for seven major vasculitic disorders through the analysis of prospectively collected patient data from 48 centers. Using two classification methods, the subcommittee derived criteria for polyareritis nodosa, Churg-Strauss syndrome, Wegner's granulomatosis, hypersensitivity vasculitis, Henoch-Schönlein purpura, giant cell (temporal) arteritis, and Takayasu's arteritis. Although such criteria may identify typical patients with a distinct form of vasculitis, they are not intended to establish a diagnosis in an individual patient; rather, they should aid comparability of different patient groups in various research endeavors.


Transport of L-glutamic acid in the fission yeast Schizosaccharomyces pombe.

Transport of L-glutamic acid into the fission yeast Schizosaccharomyces pombe grown to the early stationary phase and preincubated for 60 min with 1% D-glucose is practically unidirectional and is mediated by a single uphill transport system with a KT of 170 microM and Jmax of 4.8 nmol min-1 (mg dry wt.)-1. The system proved to be rather non-specific since all the amino acids transported into the cells acted as potent competitive inhibitors. It has a pH optimum at 3.0-4.0, the accumulation ratio of L-glutamic acid is highest at a suspension density of 0.6-1.0 mg dry wt. per ml and decreases with increasing L-glutamic acid concentrations in the external medium. The system present in the cells after preincubation with D-glucose is unstable and its activity decays after washing the cells with water or after stopping the cytosolic proteinsynthesis with cycloheximide, with a half-time of 24 min in a reaction significantly retarded by phenylmethylsulfonyl fluoride, a serine proteinase inhibitor. The synthesis of the transport protein appears to be repressible by ammonium ions.


Dietary alpha-linolenic acid deficiency in adult rats for 7 months does not alter brain docosahexaenoic acid content, in contrast to liver, heart and testes.

In adult rats, 22:6(n - 3) dietary deficiency does not affect brain membranes, but has a significant effect on some other visceral organs. 60-day-old male rats fed a diet containing sufficient amounts of both linoleic and alpha-linolenic acid were divided into three groups. One group continued the same diet; the second was fed a diet containing 2% sunflower oil, the third was fed 10% sunflower oil (sunflower oil contains linoleic acid, but trace amount of alpha-linolenic acid). Animals were killed different times after receiving the new diets (1 to 31 weeks). For animals fed the diets containing only sunflower oil, deficiency in cervonic acid content (DHA, docosahexaenoic acid, 22:6(n - 3)) was not detected in whole brain, myelin or nerve endings within 31 weeks. In contrast, this acid progressively declined in liver, heart and testes up to 3 weeks and remained nearly stable thereafter. In parallel to the reduction of cervonic acid content, 22:5(n - 6) content increased in liver and heart, but not in testes. It also increased in brain, nerve endings and myelin from week 3, 6 and, 9 respectively. These results suggest that brain cervonic acid is highly preserved or is maintained at the expense of other organs.


Role of adrenergic and cholinergic mediators in salivary phospholipids secretion.

The influence of adrenergic and cholinergic mediators on phospholipid secretion in rat sublingual salivary gland cells maintained in the presence of [3H]choline was investigated. The secretion of [3H]choline-containing phospholipids over 30 min period averaged 1.93% of the total cellular labeled phospholipids in the absence of any mediator, and was enhanced by beta-adrenergic agonist, isoproterenol, to a greater extent than the cholinergic agonists, pilocarpine and carbachol. A 2.9-fold increase in phospholipid secretion occurred with isoproterenol, while pilocarpine and carbachol evoked only 1.3-fold increase. The effect of isoproterenol was inhibited by alprenolol and that of pilocarpine and carbachol by atropine. In contrast to pilocarpine and carbachol, the enhanced phospholipid secretion due to isoproterenol was accompanied by an increase in cAMP concentration. The secretion of phospholipids was also stimulated by dibutyryl-cAMP and the protein kinase C activator, phorbol myristate acetate, but not by 4 alpha-phorbol 12,13-didecanoate which does not activate protein kinase C. Furthermore, the effects of dibutyryl-cAMP and phorbol myristate acetate were additive. The phospholipids secreted in response to isoproterenol exhibited a 52% decrease in lysophosphatidylcholine, while those secreted in response to pilocarpine and carbachol showed a 21-23% lower content of phosphatidylcholine, and were enriched in lysophosphatidylcholine (2.6-2.8-fold) and sphingomyelin (1.5-1.6-fold). The results indicate that salivary phospholipid secretion remains mainly under beta-adrenergic regulation, while the phospholipid makeup of the secretion is under cholinergic control.


Identification of pristanoyl-CoA oxidase as a distinct, clofibrate non-inducible enzyme in rat liver peroxisomes.

In this paper we describe the identification of pristanoyl-CoA oxidase activity in rat liver peroxisomes. This activity was not stimulated by clofibrate feeding. Furthermore, the activity was found in multiple tissues. These results show that pristanoyl-CoA oxidase is different from any of the known oxidases which include a clofibrate-inducible acyl-CoA oxidase and the recently identified cholestanoyl-CoA oxidase. Gelfiltration and chromatofocusing experiments provide conclusive evidence that we are dealing with a novel acyl-CoA oxidase with a unique function in peroxisomal beta-oxidation.


Cloning and nucleotide sequence of the Brucella abortus groE operon.

The cloning and sequencing of the Brucella abortus groES and groEL genes are reported. The genes are adjacent on the Brucella chromosome, and presumably comprise a functional operon. Putative promoter and terminator sequences are also identified. The groES gene exhibits 60%, and the groEl gene 69%, sequence identity with the corresponding Escherichia coli genes.


Cloning of HSP60 (GroEL) operon from Clostridium perfringens using a polymerase chain reaction based approach.

Using degenerate oligonucleotide primers for conserved regions of HSP60, a 0.6 kilobase fragment of Clostridium perfringens DNA was amplified by the polymerase chain reaction. The amplified fragment was used as a probe to isolate a genomic clone containing the C. perfringens HSP60 operon. The clone contained two open reading frames homologous to the GroES and GroEL (or HSP60) family of bacterial and eukaryotic proteins as well as other upstream and downstream sequences. The approach described here, employing this set of degenerate oligonucleotide primers, could be used to clone HSP60 gene/cDNA from any species.


Pathogenesis of peritoneal fibrosing syndromes (sclerosing peritonitis) in peritoneal dialysis.

Drawing from diverse sources including epidemiological and clinical data, surgical observations, histopathology, serosal healing responses to fibrin and fibrinolysis, tissue reaction to chronic exposure, and to exo- and endotoxins, new information on mesothelial stem cells, autocrine and paracrine influences on their proliferation and collagen synthesis, and the effect of glucose on fibroconnective tissue, we have begun to piece together the pathogenetic jigsaw of fibrosis in continuous ambulatory peritoneal dialysis (CAPD). The reaction of peritoneal mesothelium and stroma to the stress of continual dialysis results in a spectrum of alterations ranging from opacification through a tanned peritoneum syndrome to sclerosing encapsulating peritonitis (SEP). Any agent that causes irritation of the mesothelial layer and induces serositis, or single severe or multiple episodes of peritonitis resulting in mesothelial loss, predisposes the peritoneum to fibroneogenesis. An accurate definition of the histopathological changes of peritoneal thickening is a prerequisite for defining pathogenesis. This paper is the first attempt to create such a framework. It is evident from many areas of study that fibrin deposition and fibrinolysis, hyalinization of the superficial stromal collagen possibly tanned through nonenzymatic glycosylation by dialysate glucose and the proliferative potential of mesothelial stem cells play an important and possibly interdependent role in excessive fibroneogenesis in certain patients on CAPD. Many of the pieces of the jigsaw are obviously still missing, and the picture is most surely incomplete. Nevertheless, the outline of the pathologic and etiologic landscape should now be discernible.


Review article: maintenance treatment with H2-receptor antagonists for peptic ulcer disease.

In recent years a number of different strategies for managing patients with peptic ulcer disease have become available. The present review discusses the relative merits of each form of treatment. Intermittent treatment (whether given in response to symptoms or as a prophylactic regimen prescribed seasonally or at weekends) fails to prevent ulcer recurrence and leaves patients at risk of haemorrhage and perforation. Anti-Helicobacter pylori therapy, although useful in certain circumstances, cannot be recommended for all patients with ulcer disease because of side effects and, in any case, requires further assessment of efficacy. Gastric surgery reduces ulcer recurrence and complications, but operations which have a low incidence of side effects are associated with higher rates of ulcer recurrence, particularly when patients are followed up for more than 10 years. Long-term continuous maintenance treatment with H2-receptor antagonists for 5 or more years effectively prevents ulcer recurrence in the majority of patients and significantly reduces the risk of ulcer complications. In addition, maintenance treatment has proved to be safe and is well tolerated by patients. Maintenance treatment with H2-receptor antagonists is the preferred option for the management of patients with peptic ulcer disease.


Clinical tolerance to three 5-aminosalicylic acid releasing preparations in patients with inflammatory bowel disease intolerant or allergic to sulphasalazine.

The clinical tolerance to three 5-aminosalicylic acid (5-ASA) releasing preparations (mesalazine, olsalazine and balsalazide) was assessed in a consecutive series of 43 patients with inflammatory bowel disease who were intolerant to sulphasalazine. The relative contributions to the side-effects of sulphasalazine made by its two components, 5-ASA and sulphapyridine, were also assessed in these patients. Thirty-nine (91%) patients were able to tolerate at least one of the three 5-ASA preparations. Only four (9%) patients were intolerant to all preparations, having adverse reactions previously experienced with sulphasalazine and presumably related to 5-ASA rather than sulphapyridine. The clinical tolerance to mesalazine (63%), olsalazine (70%) and balsalazide (70%) was similar, and tolerance to one drug only was found in nine (18%) patients. The commonest adverse reactions associated with 5-ASA preparations were gastrointestinal. Diarrhoea was a problem in five patients during treatment with olsalazine and three each while on mesalazine and balsalazide. Allergic reactions from 5-ASA preparations were uncommon; of ten patients with rash following sulphasalazine only one developed a rash with mesalazine. The results of this study indicate that the vast majority of patients with inflammatory bowel disease can be managed with at least one of these four 5-ASA containing preparations and that the side-effects of sulphasalazine are multifactorial in aetiology, some being due to the parent molecule, and some to one of its two metabolites, 5-ASA and sulphapyridine.


The locus Om, responsible for the DDK syndrome, maps close to Sigje on mouse chromosome 11.

The DDK inbred strain of mouse has a striking particularity: when DDK females are crossed to males of other strains they exhibit a reduced fertility, whereas the reciprocal crosses (non-DDK females x DDK males) are fertile (Wakasugi et al. 1967; Wakasugi 1973). The low fertility results from an early embryonic lethality, the F1 embryos dying near the late morula-early blastocyst stage. Genetic analyses (Wakasugi 1974) and nuclear and cytoplasmic transfers (Renard and Babinet 1986; Babinet et al. 1990; Mann 1986), have shown that the failure of the embroys to develop is due to an incompatibility between a DDK maternally encoded cytoplasmic product and the non-DDK paternal genome. In order to elucidate the genetic determinism of this embryonic lethality, we have analyzed the fertility of male progeny from a backcross BALB/c females x (BALB/c x DDK)F1 males and that of males from a set of recombinant inbred (RI) strains, established from DDK and BALB/c progenitors, when mated with DDK females. Our results indicate that a single locus, Om, is responsible for the DDK syndrome and is located on Chromosome (Chr) 11, very close to the Sigje locus.


Multiple sites of crossing over within the Eb recombinational hotspot in the mouse.

The Eb gene of the mouse major histocompatibility complex (MHC) contains a well-documented hotspot of recombination. Twelve cases of intra-Eb recombination derived from the b, d, k and s alleles of the Eb gene were sequenced to more precisely position the sites of meiotic recombination. This analysis was based on positioning recombination breakpoints between nucleotide polymorphisms found in the sequences of parental haplotypes. All twelve cases of recombination mapped within the second intron of the Eb gene. Six of these recombinants, involving the k and s haplotypes, mapped to two adjoining DNA segments of 394 and 955 base pairs (bp) in the 3' half of the intron. In an additional two cases derived by crossing over between the d and s alleles, breakpoints were positioned to adjoining segments of 28 and 433 bp, also in the 3' half of the intron. Finally, four b versus k recombinants were mapped to non-contiguous segments of DNA covering 2.9 kb and 1005 bp of the intron. An analysis of the map positions of crossover breakpoints defined in this study suggests that the second intron of the Eb gene contains a recombinational hotspot of approximately 800-1000 bp which contains at least two closely linked recombinationally active sites or segments. Further examination of the sequence data also suggests that the postulated location for the recombinational hotspot corresponds almost precisely to an 812 bp sequence that shows nucleotide sequence similarity to the MT family of middle repetitive DNA.


Localization of growth arrest-specific genes on mouse chromosomes 1, 7, 8, 11, 13, and 16.

Growth arrest in NIH3T3 cells is associated with increased expression of a variety of mRNAs, several of which have been isolated as cDNA clones. Six of these growth arrest-specific (Gas) genes were mapped by following the inheritance of DNA restriction fragment length variants (RFLVs) associated with them in panels of recombinant inbred (RI) strains of mice and in the progeny of backcrosses both between laboratory mouse strains and between a laboratory strain and Mus spretus. The six genes are unlinked. Gas-1 maps to Chromosome (Chr) 13, Gas-2 to Chr 7, Gas-3 to Chr 11, Gas-4 to Chr 16, Gas-6 to Chr 8, and Gas-10 to Chr 1.


A strategy for rapid production and screening of yeast artificial chromosome libraries.

We describe methods for rapid production and screening of yeast artificial chromosome (YAC) libraries. Utilizing complete restriction digests of mouse genomic DNA for ligations in agarose, a 32,000-clone library was produced and screened in seven weeks. Screening was accomplished by subdividing primary transformation plates into pools of approximately 100 clones which were transferred into a master glycerol stock. These master stocks were used to inoculate liquid cultures to produce culture "pools," and ten pools of 100 clones were then combined to yield superpools of 1,000 clones. Both pool and superpool DNA was screened by polymerase chain reaction (PCR) and positive pools representing 100 clones were then plated on selective medium and screened by in situ hybridization. Screening by the two tiered PCR assay and by in situ hybridization was completed in 4-5 days. Utilizing this methodology we have isolated a 150 kb clone spanning the alpha 1(I) collagen (Col1a1) gene as well as 40 kb clones from the Hox-2 locus. To characterize the representation of the YAC library, the size distribution of genomic Sal I fragments was compared to that of clones picked at random from the library. The results demonstrate significant biasing of the cloned fragment distribution, resulting in a loss of representation for larger fragments.


Genomic organization and nucleotide sequence of a long mosaic repetitive DNA in the mouse genome.

A long mosaic repetitive sequence (LMRS) was isolated from a mouse liver genome library using a mouse repetitive DNA as a probe. LMRS exhibits the following features: (1) it is almost 15 kb in length; (2) it is partly organized in tandem array and frequently interrupted by other repeated sequences; and (3) it is located predominantly on the A3 band of the mouse X Chromosome (Chr). One fragment of LMRS (B6) shows restriction fragment length polymorphism (RFLP) between different mouse strains, and is thus potentially useful for mapping studies. The nucleotide sequence confirms a mosaic organization of LMRS which includes three repeats in the 5' part, showing similarity with the 5' end of L1Md-A2, and seven long A + T rich segments in the central part of the element. Our findings suggest that this sequence may have arisen from the duplication of an ancestral motif and has expanded by successive waves of amplification and invasion by foreign sequences.


Mapping of the mouse ornithine decarboxylase-related sequence family.

A family of DNA sequences homologous to the mRNA encoding ornithine decarboxylase (ODC) and comprising approximately 12 members in the mouse genome has been analyzed genetically. The inheritance of variant DNA restriction fragments detected by ODC cDNA probes on Southern blots of DNA from inbred strain mice was determined in six sets of recombinant inbred (RI) mouse strains. The distributions of these variations among the RI strains were then compared with the RI strain distribution patterns (SDPs) of previously mapped loci. This allowed the identification of nine independent ODC-related loci, of which eight could be localized to specific regions of the mouse genome: Odc-rs1 near Lamb2 on Chromosome (Chr) 1; Odc-rs2 near Psp on Chr 2; Odc-rs5, a complex locus comprising at least 5-7 copies of the ODC sequence, associated with Igk on Chr 6; Odc-rs6 between Abpa and Tam-1 on proximal Chr 7; Odc-rs7 near Hbb on distal Chr 7; Odc-rs12 near Agt and Emv-2 on distal Chr 8; Odc-rs8 associated with the Igh complex on Chr 12; and Odc-rs9 near Otf-3f on Chr 14. The ODC-related sequence family thus comprises a set of genomically dispersed "marker" loci, and alleles for several of these loci can be analyzed simultaneously in DNA from mice or cell lines. DNA from mice of 70 inbred strains has been characterized for alleles at all nine Odc-rs loci.


Factors affecting blood stem cell collections following high-dose cyclophosphamide mobilization in lymphoma, myeloma and solid tumors.

Sixty patients with malignancy were enrolled in a study of high-dose chemotherapy and peripheral blood stem cell transplantation (PBSCT). Stem cells were harvested prior to PBSCT using high-dose cyclophosphamide (CY) mobilization (4 or 7 g/m2) with collection of a median of 4.6 x 10(8)/kg mononuclear cells (range 0.2-9.5) and 21.6 x 10(4)/kg colony forming unit-granulocyte/macrophage (CFU-GM) (range 0.1-220). Forty-seven patients were mobilized once, 11 required two cycles and two required three cycles. Eight patients (13%) failed to reach the optimum CFU-GM target (greater than 15 x 10(4)/kg) following CY mobilization. A number of factors identified those patients who were likely to achieve optimum CFU-GM collections with CY mobilization. These included the use of the higher CY mobilization dose, a longer interval from last chemotherapy cycle to mobilization, and a higher premobilization bone marrow CFU-GM level. Patient's age, the degree of bone marrow infiltration, the nature of disease or the number of pre-mobilization chemotherapeutic cycles did not affect the ability to collect optimum CFU-GM numbers. Whilst the mobilization procedure was associated with moderate non-hematologic toxicity, significant hematological morbidity was observed primarily in patients mobilized using the 7 g/m2 dose. Refinements to the protocol, in particular the use of hematopoietic growth factors, are currently under investigation.


Tardive dyskinesia. Patients' lack of awareness of movement disorder.

Of 113 patients in long-stay wards of a psychiatric hospital, 43 had TD. Twenty-six of the 39 patients who consented to take part in the study were unaware of abnormal involuntary movements. These patients scored significantly lower on a short test of cognitive function than patients who were aware of such movements. The diagnosis of schizophrenia, particularly the 'defect' state with cognitive deficit and negative symptoms, was found to be associated with lack of awareness of TD.


Spouse-aided therapy with agoraphobics.

Sixty agoraphobics were treated by behavioural therapy (self-exposure in vivo) either with their partner involved in all aspects of treatment or without their partner. The two treatment formats were about equally effective. Behavioural treatment directed at the agoraphobia resulted in improvement irrespective of marital quality and partner involvement in the therapy. The effects of treatment led neither to a deterioration of the marriage nor to adjustment problems in the partner. Avoidance behaviour, intropunitivity and overprotection were found to predict treatment response. The partners of agoraphobics were not found to have psychological problems themselves.


A prospective study of panic and anxiety in agoraphobia with panic disorder.

The features of panic and anxiety in the natural environment were studied by prospective self-monitoring in 39 patients with chronic agoraphobia and panic disorder. Panics overlapped greatly with anxiety episodes but were more intense. Panics occurred more often in public places than did anxiety episodes, but had otherwise similar symptom profile, time of occurrence, and antecedents. Most panics surged out of a pre-existing plateau of tonic anxiety which lasted most of the day. Spontaneous panics were less frequent than situational panics and occurred more often at home but were otherwise similar. These findings do not support the sharp distinction between panic and anxiety in DSM-III-R, not its emphasis on spontaneous panic in classifying anxiety disorders. Thoughts of dying and 'going crazy'/losing control accompanied only a minority of panic/anxiety episodes and seemed to be a product of intense panic rather than a cause.


Cell proliferation in childhood acute leukemia. Comparison of Ki-67 and proliferating cell nuclear antigen immunocytochemical and DNA flow cytometric analysis.

The proliferative activity of bone marrow leukemia cells was determined by DNA flow cytometric (FCM) analysis and labeling index (LI) of Ki-67 monoclonal antibodies and proliferating cell nuclear antigen (PCNA) autoantibodies in 73 children with acute leukemia. LI of Ki-67 varied greatly from patient to patient (range, 0.4% to 42.2%; mean, 18.8%) and differed significantly between acute lymphoblastic leukemia (ALL) and acute nonlymphoblastic leukemia (ANLL). In ALL, the Ki-67 LI showed a positive correlation with the S-phase fraction (SPF) determined by DNA FCM analysis, whereas, in ANLL, there was a discrepancy between the Ki-67 LI and SPF. In contrast, LI of PCNA varied less among the patients (range, 57.2% to 100%; mean, 90.3%), and the value was always higher than that of the Ki-67 LI in individual patients. A significant relationship between PCNA LI and the percentage of blast cells was found in peripheral blood leukocytes from patients with leukemia. These results suggest that the Ki-67 LI reflects differences in the proliferative activity depending on the subtype of the disease and that the PCNA LI is useful as a marker of proliferating cells.


Internal mammary artery angiography should be a routine component of diagnostic coronary angiography.

Left internal mammary artery (LIMA) angiography was performed with diagnostic coronary angiography in 130 cases for which the coronary findings made use of the LIMA as a bypass graft a consideration. In 98% of the cases the approach to LIMA angiography was femoral with a 5F LIMA catheter first directed into the proximal subclavian and then advanced over a guidewire placed into the distal subclavian well beyond the origin of the LIMA. After withdrawing the wire the catheter was brought proximally to selectively cannulate and visualize the LIMA with nonionic contrast media. The only complication was a single transient occipital visual field loss. LIMA caliber too narrow to permit use as a graft was found twice, LIMA occlusion unrelated to prior surgery was found once, and LIMA occlusion related to prior surgery was found twice. Subclavian and/or vertebral stenosis was present five times. Large proximal branches of the LIMA best identified prior to surgery were present 12 times. Based on this experience, LIMA angiography 1) can be performed safely with a high degree of success, 2) demonstrates significant findings in 15% of cases, and 3) should therefore be performed whenever coronary angiographic findings make it appropriate to consider LIMA to coronary artery bypass grafting.


Modulation of adhesion molecules on human large granular lymphocytes by interleukin-2 in vivo and in vitro.

The modulation of adhesion molecules on human large granular lymphocytes (LGL) by interleukin (IL)-2 was investigated both in vivo and in vitro. Intercellular adhesion molecule-1 (ICAM-1; CD54) expression increased on LGL of cancer patients receiving IL-2 adoptive immunotherapy. ICAM-1 expression on LGL isolated by Percoll gradient centrifugation, LGL purified, and expanded by adherence to plastic surfaces and LGL identified by Leu 19 (CD56) monoclonal antibody were increased significantly in response to IL-2 in vitro. Exposure of LGL to IL-1, interferon (IFN)-gamma, and tumor necrosis factor (TNF) in vitro did not induce ICAM-1. The expression of LFA-1 (CD11a/CD18), a receptor for ICAM-1, and other leukocyte adhesion molecules, including Mac-1 (CD11b/CD18) and p150,95 (CD11c/CD18), was only maintained by IL-2. IL-2 induction of ICAM-1 and the maintenance of CD18 complex expression on small lymphocytes separated by Percoll gradients were similar to that on LGL. We conclude that IL-2 enhances the expression of ICAM-1 on multiple human lymphocyte populations including LGL effectors. Expression of the CD18 complex on LGL does not appear to be highly regulated by IL-2. These findings may have implications relevant to the role of these adhesion molecules in the activities of LGL modulated by IL-2.


Mechanism of pokeweed mitogen inhibition of rhIL-4-induced human IgE synthesis.

Pokeweed mitogen (PWM) suppressed rhIL-4-induced IgE synthesis in a concentration-dependent manner. When rhIL-4 was present from Day 0, PWM added to cultures on Day 0 or 3 inhibited MNC IgE synthesis but not when it was added on Day 6 or later. The concentration of interferon-gamma (IFN-gamma) in MNC culture supernatants varied directly with the quantity of PWM added. Conversely, rhIL-4-stimulated MNC culture IgE concentrations varied inversely with the dose of PWM added and the IFN-gamma concentrations induced. The addition of a rabbit polyclonal neutralizing anti-human IFN-gamma antibody to rhIL-4 plus PWM-stimulated cultures partially or completely reversed PWM-induced inhibition of rhIL-4-induced IgE synthesis. PWM failed to inhibit rhIL-4-induced IgE synthesis by isolated B cells cocultured with monocytes and T cells from a clone unable to produce IFN-gamma message or protein. These findings are consistent with the postulate that PWM inhibits rhIL-4-induced IgE synthesis by inducing the production of IFN-gamma.


Suppression of collagen arthritis with antibodies to an arthritogenic, oligoclonal T cell line.

Rats immunized with type II collagen (CII) develop an immunologically mediated polyarthritis. T cells have been implicated in the pathogenesis of this model since they can adoptively transfer the disease. A CII-specific T cell line (VA), consisting of three distinct clones by Southern blot analysis, has been shown to be arthritogenic. Antibodies specific for this line were generated by immunizing rabbits. In an attempt to prevent collagen-induced arthritis (CIA), Louvain rats were injected with 1 ml of anti-VA ip on Days -1, +1, +3 and 0.5 ml on Day +5 (early treatment). To evaluate its effect on existing disease, rats received anti-VA on the day of arthritis onset and subsequently on 4 successive alternate days using the same dosage protocol (late treatment). Control rats received no therapeutic injections or were administered normal rabbit serum. All rats were immunized with CII on Day 0 to induce CIA. Rats administered antibodies using the early anti-VA treatment protocol had a significantly diminished incidence of arthritis compared to controls. Established arthritis was significantly diminished compared to controls in rats given the late anti-VA treatment. In both protocols, radiographic evidence of joint destruction was significantly reduced compared to controls. T cell phenotyping using flow cytometry analysis demonstrated that the anti-VA antibody therapy selectively eliminated a small subset of T cells since there was little difference in total T cell counts in the experimental versus control groups. Delayed type hypersensitivity and IgG antibody titers to CII were minimally decreased in the experimental versus control group. These results suggest that antibodies raised to an oligoclonal arthritogenic T cell line can suppress collagen arthritis. This may have implications with respect to 1) the size of the T cell receptor repertoire involved in the pathogenesis of collagen arthritis and 2) immunospecific protocols for CIA and other autoimmune diseases.


Isolation and characterization of an IL-1-dependent IL-4-producing bovine CD4+ T cell clone.

An Ag-specific interleukin 1 (IL-1)-dependent bovine CD4+ Th cell clone, termed 300B1, was isolated and found to resemble the previously described IL-1-dependent murine CD4+ Th2 cell clone, D10.G4.1. Both the 300B1 and the D10.G4.1 T cell clones proliferated to bovine (Bo) IL-1 beta, human (Hu) IL-1 alpha and IL-1 beta, and murine IL-1 alpha when cells were costimulated with concanavalin A (Con A). Proliferation of the 300B1 clone, when costimulated with Con A, appeared to be IL-1-specific in that proliferation could not be promoted by BoIL-2, HuIL-3, HuIL-4, HuIL-5, or HuIL-6. The 300B1 clone produced interferon-gamma (IFN-gamma), but not IL-2 following stimulation with either Con A, Con A plus phorbol 12-myristate 13-acetate or Ag plus antigen-presenting cells. Upon stimulation with Con A, the 300B1 clone expressed IL-4 mRNA and produced an autocrine growth factor (AGF) that could be inhibited by anti-HuIL-4 but not by anti-HuIL-2 Ab. The clonal derivation of the 300B1 clone was confirmed by isolating five 300B1 subclones, all of which produced IFN-gamma and an AGF but not IL-2. Collectively, these results suggest the IL-1-dependent bovine 300B1 Th cell clone produces IL-4, but not IL-2, as an AGF. Furthermore, the bovine Th cell clone appeared to share many characteristics of previously described murine Th2 cell clones except that the bovine clone produced IFN-gamma.


Onset of vecuronium neuromuscular block is more rapid in patients undergoing caesarean section.

This investigation was carried out in ten patients undergoing elective Caesarean section and the results were compared with those of a control group of ten nonpregnant females of the same age group. The study investigated the onset of vecuronium neuromuscular block and the conditions of tracheal intubation when ketamine (1.5 mg.kg-1)-vecuronium 100 micrograms.kg-1) sequence was used for rapid-sequence induction of anaesthesia. The ulnar nerve was stimulated supra-maximally at the wrist with train-of-four stimuli every 20 sec, and the electromyographic response of the adductor pollicis muscle was displayed. The onset of 50% neuromuscular block as monitored by electromyography was shorter in the Caesarean group (80 +/- 30 sec) than in the control group (144 +/- 43 sec). The conditions of intubation at 50% block were adequate in both groups. Also, the onset of 90% block was shorter in the Caesarean group. The time of recovery to T1/control ratio of 25% was longer in the Caesarean group (46 +/- 10 min) than in the control patients (28 +/- 10 min). The results show that administration of vecuronium according to body weight results in a more rapid onset and delayed recovery of neuromuscular block in pregnant women undergoing Caesarean section than in the nonpregnant control patients.


No effect of a Taq1 polymorphism in DNA at the endothelin I (EDN1) locus on normal blood pressure level or variability.

Endothelin is a peptide reported to be one of the most potent vasoconstrictors known. Presumably, endothelin could play a role in the physiological regulation of blood pressure in healthy or hypertensive people. We have studied a normal restriction fragment length polymorphism (RFLP) at the endothelin-I (EDN1) locus detected with the restriction enzyme TaqI. In three different series comprising 166, 120 and 207 unrelated individuals, we found no evidence for association between genotype in this polymorphism and level of systolic or diastolic blood pressure. In two series of 156 and 117 monozygotic (MZ) twin pairs, respectively, there was no difference between genotypes in within-pair variation in systolic or diastolic blood pressure. Thus neither "level gene" nor "variability gene" effects of normal genes at the EDN1 locus could be detected with the polymorphism analyzed, in healthy population samples.


Combined total deficiency of C7 and C4B with systemic lupus erythematosus (SLE).

The first inherited combined total deficiency of C7 and C4B complement components associated with SLE is described in a young female. Functional C7 assays showed a homozygous C7 deficiency in the propositus and her sister, and an heterozygous one in their parents. C4 molecular analyses showed that both the propositus and her mother had two HLA haplotypes carrying only C4A-specific DNA sequences and a normal C4 gene number. Thus, only C4A proteins could be expressed, with resultant normal C4 serum levels. The coexistence of a combined complete C7 and C4B deficiency may therefore abrogate essential functions of the complement cascade presumably related to immune complex handling and solubilization despite an excess of circulating C4A. These findings challenge the putative pathophysiological roles of C4A and C4B and stress the need to perform both functional assays and C4 allotyping in patients with autoimmune pathology and low haemolytic activity without low serum levels of a classical pathway complement component.


Evidence for the presence of activated CD4 T cells with naive phenotype in the peripheral blood of patients with rheumatoid arthritis.

We have investigated whether T cell activation in rheumatoid arthritis (RA) preferentially engages distinct T cell subpopulations in the peripheral blood (PB) and in the synovial fluid. We found that CD25 expression was enhanced among PB CD4 T cells of RA patients as compared with CD4 cells of patients with reactive arthritis, degenerative joint disease or of healthy controls. Within the CD4 T lymphocytes subset we found that the CD45RO- (naive) cells selectively in RA displayed higher levels of CD25 protein and of interferon-gamma mRNA expression when compared with the respective subset of all other investigated groups. These results show that in the PB of RA, but not in the PB of the other arthropathies or healthy controls, CD45RO-CD4 T lymphocytes exist which display well-defined signs of activation.


Decreases in alpha beta T cell receptor negative T cells and CD8 cells, and an increase in CD4+ CD8+ cells in active Hashimoto's disease and subacute thyroiditis.

We examined peripheral lymphocyte subsets in patients with autoimmune thyroid disease, or subacute thyroiditis, in the active stage when possible. During destructive thyrotoxicosis arising from alpha beta T cell receptor (TCR) negative T (WT31-CD3+) cells and CD8 (CD4-CD8+) cells decreased and those of CD4+CD8+ cells increased slightly, resulting in proportional increases in CD4 (CD4+CD8-) cells, non-T, non-B (CD5-CD19-) cells, and the CD4/CD8 cell ratio. Changes were similar in active subacute thyroiditis. During stimulative thyrotoxicosis in active Graves' disease, the numbers of such T lymphocyte subsets were not changed, but only the number of CD5+ B (CD5+CD19+) cells increased markedly, resulting in proportional decreases in total T (CD3+) cells, alpha beta+ TCR T (WT31+CD3+) cells, CD8 cells, and non-T, non-B cells. A serial study of some of the patients showed opposite changes in alpha beta TCR- T cells, the CD4/CD8 cell ratio, and CD5+ B cells between the active stages of Graves' and Hashimoto's diseases. alpha beta TCR- T cells were mostly gamma delta TCR+ T (IIF2+ CD3+) cells in these patients. These data suggest that alpha beta TCR-T (gamma delta TCR+ T), CD8, and CD4+ CD8+ cells are important in thyroid destruction in Hashimoto's disease and subacute thyroiditis, and that CD5+ B cells are important in thyroid stimulation in Graves' disease.


Suppression of efferent limb of testicular autoimmune response by a regulatory CD4+ T cell line in mice.

A murine T cell line (designated as C.Ts) as a mediator of suppression of experimental autoimmune orchitis (EAO) was established. The method of establishment of C.Ts cell line was preparing spleen cells from C3H/He mice hyperimmunized with testicular germ cells (TC) and the repeated selection of the lymphocytes in vitro by stimulation with mouse testicular antigens (mTA). The C.Ts cells were Thy1.2+, surface immunoglobulin-, CD3+, CD4+ and CD8-. The cells could suppress the induction of EAO when transferred into actively EAO-sensitized mice only at the pre-clinical stage of the disease (efferent limb of the autoimmune response). The transferred C.Ts cells significantly inhibited both cellular and humoral immune responses to TC in the recipients in an antigen-specific manner. The disease suppression by C.Ts cells was found to depend upon their cell number, and their suppressive activity was markedly augmented by in vitro stimulation with mTA.


The human catechol-O-methyltransferase (COMT) gene maps to band q11.2 of chromosome 22 and shows a frequent RFLP with BglI.

We have been able to assign the human catechol-O-methyltransferase gene (COMT) to chromosome 22q11.2 by using Southern blot analysis of panels of somatic cell hybrids and chromosomal in situ hybridization. Furthermore, Southern blot analysis of DNA from blood and bone marrow samples of a patient with chronic myeloid leukemia (CML), having an extra Philadelphia chromosome (Ph1) in addition to the one produced by the reciprocal translocation between chromosomes 9 and 22, showed increased COMT and BCR gene dosage as compared to DNAs originating from CML patients with only one Ph1 chromosome or from chromosomally normal individuals. Control hybridizations of the same blot with TCRG- and TCRA-specific probes showed corresponding signal intensities in all samples. A relatively frequent two-allele COMT gene RFLP (PIC = 0.37) was recognized in DNAs digested with BglI. Our gene mapping result is in concordance with that previously reported by Brahe et al. (1986), who used an autoradiozymogram assay on different somatic cell hybrids to map this gene to chromosome 22.


The role of dopamine in mood disorders.

The findings on dopamine in mood disorders suggest that decreased dopamine activity is involved in depression, while increased dopamine function contributes to mania. This report reviews the considerable preclinical and clinical evidence supporting this hypothesis, with particular emphasis on specific subtypes of depression. We also discuss the importance of integrating these dopamine findings with dopamine brain circuitry and with other neurotransmitter theories of affective disorders.


[Acquired protein C deficiency in ulcerative colitis. The cause of thromboembolic complications].

Three patients with an acute exacerbation of ulcerative colitis (a 40-year-old and a 31-year-old man and a 30-year-old woman) developed a protein C deficiency (serum protein C activity between 32 and 48%). In the two men the protein C deficiency was diagnosed only after the onset of severe thromboembolic complications (cavernous sinus thrombosis; pulmonary embolism) during heparin treatment. But in the woman protein C activity was measured immediately after hospital admission (in the knowledge of the first two cases) even before heparin administration was started. All three patients received treatment with sulphasalazine (3 g daily) and fluocortolone (60 mg daily), as well as full heparinization (22,500-36,000 IU daily). Protein C activity returned to normal on remission of the ulcerative colitis (in one case only after subtotal colectomy). These case reports show that acquired protein C deficiency can be reversed by rigorous treatment of the underlying disease.


Permeability of bovine brain microvessel endothelial cells in vitro: barrier tightening by a factor released from astroglioma cells.

It has been shown both in vivo and in culture that astrocytes communicate with brain microvessel endothelial cells (BMECs) to induce many of the blood-brain barrier characteristics attributed to these unique cells. However, the results using cultured cells are conflicting as to whether this communication is dependent upon cell-cell contact. In this study we used primary cultures of bovine BMECs grown as monolayers on polycarbonate filters to study the formation of the barrier in vitro and examine its modulation by rat C6 glioma cells. Effects were examined by treating postconfluent BMEC monolayers with medium conditioned continually by C6 cells from the basolateral side to mimic the in vivo orientation. Cell monolayer integrity was assessed using electrical resistance and by measuring diffusion of uncharged molecules. BMEC monolayers form a functionally polarized and leaky barrier, with maximal resistance of 160 omega . cm2 and significant flux of molecules of molecular weight less than 350 Da. Treatment with rat or human astroglioma cells rather than pericytoma cells or transformed fibroblasts results in a concentration-dependent 200-440% increase in electrical resistance and a coincident 50% decrease in permeability to sucrose and dextran (70 kDa). The decrease in passive diffusion is most likely due to a change in tight junctions and not to transcellular vesicular traffic. The findings support that astroglioma cells release one or more signals that are required for cultured BMECs to express a "differentiated" phenotype associated with a tighter barrier, increased gamma-glutamyl transpeptidase activity, and decreased pinocytic activity. The relative ease and quickness of this culture system makes it amenable to studies on cell-cell interaction and regulation of barrier maintenance.


Neuropsychological test scores and clinical response to neuroleptic drugs in schizophrenic patients.

Schizophrenic patients who showed greater neuropsychological deficits on the Luria-Nebraska Pathognomonic subscale (PATH) and Trail-Making Test showed less improvement during treatment with neuroleptic drugs than patients with lower scores on these neuropsychological tests. Other neuropsychological test scores we used were not consistently related to the patients' degree of clinical improvement during neuroleptic treatment. Only a few patients evidenced severe scores on either the PATH or TM tests, suggesting that more subtle neuropsychological deficits may identify a subgroup of schizophrenics who will show a relatively poor response to treatment with standard neuroleptic drugs.


Increased tardive dyskinesia in alcohol-abusing schizophrenic patients.

Many schizophrenics have a diagnosis of substance abuse or dependence. We evaluated whether drug or alcohol abuse is an independent risk factor for tardive dyskinesia (TD) in schizophrenia. In a consecutive admission, clinical study of 75 hospitalized schizophrenics, drug or alcohol abusers had significantly higher TD scores than nonabusers. The association of alcohol abuse or dependence with TD seemed independent from other risk factors for TD.


Radioligand binding studies of the atypical beta 3-adrenergic receptor in rat brown adipose tissue using [3H]CGP 12177.

Two populations of [3H]CGP 12177 binding sites exist in rat interscapular brown adipose tissue (IBAT) plasma membranes. The majority of binding sites are of low affinity with a Kd of 31 nM, a value in close agreement with that for the Kd of [3H]CGP 12177 binding to a cloned rat beta 3-adrenergic receptor (AR) expressed in CHO cells (44 nM). Competition binding studies demonstrate that the Ki values of the cloned rat beta 3-AR and of the low affinity sites in IBAT are 45 and 29 nM, respectively, for BRL 37344 and 1.4 and 1.0 microM, for (-)-propranolol. These findings strongly suggest that the low affinity [3H]CGP 12177 binding site measured in IBAT plasma membranes represents the atypical beta 3-AR in this tissue.


Structure of holo-chaperonin studied with electron microscopy. Oligomeric cpn10 on top of two layers of cpn60 rings with two stripes each.

A structural model of holo-chaperonin, known as a protein-folding control protein comprising 60 kDa (cpn60) and 10 kDa polypeptides (cpn10), is proposed based on the electron microscopic images of holo-chaperonin from Thermus thermophilus and cpn60 from Paracoccus denitrificans. Isolated Paracoccus cpn60 shows very similar images to those of Escherichia coli tetradecameric cpn60, a seven-membered ring in the top view and a rectangular shape with four stripes in the side view. However, a small number of half-thick rectangles with two stripes are also seen which indicates that a single cpn60-heptamer ring has two stripes parallel to the plane of the ring. Thermus holo-chaperonin shows a bullet-like shape in the side view, and antibody against cpn10 binds only to the round side of the bullet. We conclude that a single cpn60-heptamer ring with two stripes stacks into two layers, and a cpn10 oligomer binds to one side of the layers.


Subcellular localisation and processing of non-specific lipid transfer protein are not aberrant in Rhizomelic Chondrodysplasia Punctata fibroblasts.

The import into peroxisomes and maturation of peroxisomal 3-oxoacyl-CoA thiolase are impaired in patients with the Rhizomelic form of Chondrodysplasia Punctata (RCDP). Here we show by means of immunoblotting and subcellular fractionation that non-specific lipid transfer protein (nsLTP), another peroxisomal protein synthesised as a larger precursor, is localised in peroxisomes and is present as the mature protein in RCDP fibroblasts. Thus the component of the import machinery defective in RCDP is not required for the import of nsLTP into peroxisomes.


Neuroactive steroids.

Neuroactive steroids are natural or synthetic steroids that rapidly alter the excitability of neurons by binding to membrane-bound receptors such as those for inhibitory and (or) excitatory neurotransmitters. The best-studied neuroactive steroids are a series of sedative-hypnotic 3 alpha-hydroxy ring A-reduced pregnane steroids that include the major metabolites of progesterone and deoxycorticosterone, 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (allotetrahydroDOC), respectively. These 3 alpha-hydroxysteroids do not interact with classical intracellular steroid receptors but bind stereoselectively and with high affinity to receptors for the major inhibitory neurotransmitter in brain, gamma-amino-butyric acid (GABA). Biochemical and electrophysiological studies have shown that these steroids markedly augment GABA-activated chloride ion currents in a manner similar (but not identical) to that of anesthetic barbiturates. Several steroids have also been observed to have convulsant or proconvulsant properties, including the synthetic amidine 3 alpha-hydroxy-16-imino-5 beta-17-azaandrostan-11-one (RU5135) and the natural sulfate esters of pregnenolone and dehydroepiandrosterone. Several of these have been shown to be bicuculline or picrotoxin-like GABAA receptor antagonists. Examples of steroids that alter neuronal excitability rapidly by augmenting or inhibiting excitatory amino acid receptor-mediated responses have also been reported. Recently, allopregnanolone and allotetrahydroDOC have also been measured in brain and plasma where their levels have been shown to fluctuate in response to stress and during the estrous and menstrual cycles of rats and humans, respectively. Although the major fraction of allopregnanolone in tissue, including brain, is of adrenal and/or ovarian origin, appreciable levels of allopregnanolone can still be measured in the brains of adrenalectomized and/or oophorectomized animals. Receptor-active neurosteroids may represent an important class of neuromodulators that can rapidly alter central nervous system excitability via novel nongenomic mechanisms.


Hemodynamic effects of terbutaline, a beta 2-adrenoceptor agonist, in conscious rats with secondary biliary cirrhosis.

The hemodynamic responses to terbutaline - a selective beta 2-adrenoceptor agonist - were studied in conscious normal rats and in conscious rats with secondary biliary cirrhosis. Compared with those of normal rats, dose-response curves in cirrhotic rats indicated significantly decreased reactivity in arterial pressure and heart rate. Half-maximal effective dose was not significantly different between the two groups. Terbutaline induced significant, dose-dependent decreases in portal pressure in both normal rats (9.3%) and cirrhotic rats (13.8%). In normal rats, terbutaline administration (32 micrograms.min-1.kg-1 body wt) increased both cardiac output and portal tributary blood flow, thus mimicking hemodynamic changes in cirrhotic rats. In cirrhotic rats, despite a significant increase in portal tributary blood flow (from 19.9 +/- 1.7 ml/min to 22.7 +/- 1.5 ml/min), terbutaline decreased portal pressure from 17.4 +/- 1.0 mm Hg to 15.0 +/- 0.8 mm Hg. This study indicates that increased beta 2-adrenoceptor stimulation in cirrhotic rats may be involved in hyperdynamic circulation. The association of a decreased portal pressure and increased splanchnic blood flow suggests that beta 2-adrenoceptor stimulation may modulate hepatic and portal collateral vascular resistance.


Induction of colitis in rats by 2-2'-azobis(2-amidinopropane) dihydrochloride.

Reactive oxygen metabolites (ROM) may play a role in the pathophysiology of inflammatory bowel disease (IBD) and ischemia-reperfusion-induced intestinal injury. Although there are many reports of intestinal mucosal injury associated with neutrophil-derived ROM, free radicals themselves have not been reported to induce intestinal mucosal injury. We administered intrarectally 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) to rats, an azo compound that generates free radicals in vitro. Acute mucosal injury was assessed histologically by light microscopy and biochemically by myeloperoxidase (MPO) activity. Intrarectal administration of AAPH (60, 90, 150 mg/kg) caused erythema, edema, and histologically verifiable mucosal inflammation. MPO activity was increased 9- to 18-fold above the control level. The levels of thiobarbituric acid (TBA) reactants and sulfhydryls (SH) were significantly (P less than 0.01) increased and decreased, respectively, by 90 mg/kg AAPH. Sulfasalazine, 5-aminosalicylic acid, the LTB4 receptor antagonist SC-41930, and the antioxidant glutathione prevented the inflammation. This model of mucosal inflammation may be useful in evaluating new therapeutic agents for the treatment of IBD.


Pattern of Sertoli cell degeneration in cryptorchid prepubertal testes.

Seventy-three testicular biopsies from 54 children (aged 2 months-14 years) with undescended testes were examined by light and electron microscopy. The biopsies included abdominal, inguinally fixed, inguinally moveable, and retractile testes. Alterations in Sertoli cell morphology were found in all biopsies. The alterations included dilated elements of rough endoplasmic reticulum, vacuolization of the cytoplasm, mitochondria with poorly preserved cristae, increase in electron density of the matrix, elongation of the nuclei, and irregularities of the nuclear membrane. According to the numerical appearance of these cells and to the extent of lesions in single Sertoli cells, seven phases in the continuous process of tubular alteration were distinguished. The most severe tubular damaged (phase VII) occurred when the seminiferous epithelium consisted exclusively of necrotic cells. All phases of tubular alterations were seen regularly in each of the biopsies investigated. Germ cells occurred only in phases I-IV and were never observed in tubules in phases V-VII. Significant differences became evident between inguinal and retractile testes by morphometric evaluation. It was demonstrated that the number of germ cells per cross-sectioned tubule (S/T value) correlated negatively with the percentage of tubules in phases V-VII. In contrast to inguinal testes, a complete absence of Sertoli cells and an S/T value less than 0.1 were never found in retractile testes and the percentage of tubules in phases V-VII was reduced significantly compared with inguinal testes. Our findings indicate that (i) maldescended testis in patients between 1 and 15 years-of-age is associated with a special pattern of Sertoli cell degeneration; (ii) Sertoli cell degeneration is a continuous process, which can lead eventually to complete dissolution of the seminiferous epithelium; (iii) total degeneration is not related to age but is dependent on testicular position; (iv) a defined phase of degeneration excludes germ cell development, and therefore enhanced Sertoli cell degeneration in cryptorchid testes must also account for the reduction in germ cell number.


Over-expression of p53 nuclear oncoprotein in colorectal adenomas.

p53 is a nuclear phosphoprotein which controls normal cell growth. Normal p53 protein is undetectable by standard immunohistochemical staining and the over-expression found in neoplastic cells correlates with the presence of point mutations of evolutionary conserved regions of the p53 gene. We examined the expression of p53 protein in a series of 36 colorectal adenomas (13 tubular, 17 tubulovillous, 6 villous) showing different degrees of dysplasia (11 mild, 19 moderate, 6 severe), 11 moderately differentiated adenocarcinomas (6 Duke's A, 4 Duke's B, 1 Duke's C) and 5 metaplastic polyps using the polyclonal antibody CM1 which recognises p53 protein in conventionally fixed and processed histological material. We found that 15 out of 36 colorectal adenomas showed p53 immunoreactivity, although in 4 positive cases (26%) the staining was very focal (less than 0.1% positive cells). More than 80% of severely dysplastic adenomas showed strong p53 immunoreactivity and this over-expression was correlated with increased cell proliferative rate as detected by the proliferating-cell-nuclear-antigen (PCNA) staining. p53 nuclear staining was also seen in 8 out of 11 (65%) colorectal adenocarcinomas as previously shown. Our data suggest that the p53 gene mutation, with the subsequent over-expression of the protein, occurs in colorectal adenomas and may therefore be a fundamental genetic event underlying the dysplasia and loss of proliferative control that are characteristic of adenomas with malignant potential.


Protein kinase C activity and expression in normal and adenomatous human pituitaries.

Protein kinase C (PKC) activity and expression were measured in 54 adenomas (prolactin (PRL)-, growth hormone (GH)- and non-secreting), 1 of them obtained from a patient treated with the dopamine agonist bromocriptine and 2 from patients treated with the somatostatin analog octreotide. They were also measured in normal human and rat pituitaries. Total PKC activity was measured by incorporation of 32P into histones, and PKC expression by dot blot immunoquantification using purified PKC as a standard. Both enzyme activity and expression were higher in adenomatous pituitaries than in normal human or rat pituitaries. PKC expression in GH-secreting and non-secreting tumors was significantly higher than that in PRL-secreting tumors. Furthermore, it was significantly higher invasive tumors than in non-invasive tumors. In the 3 adenomas which were obtained from patients treated with bromocriptine or octreotide and which were used for PKC-activity measurement, particulate- and soluble-PKC activities were significantly lower than those measured in non-treated adenomas.


Basic fibroblast growth factor induces proliferation of a rat pancreatic cancer cell line. Inhibition by somatostatin.

AR4-2J, a rat pancreatic acinar-tumor cell line, was used to investigate long-term effects of basic fibroblast growth factor (bFGF) and somatostatin on pancreatic cancer cells. We observed that bFGF stimulated cell proliferation when cells were cultured in serum-free medium. The effect was dose-dependent with half-maximal and maximal effects at 25 pM and I nM bFGF, respectively. The somatostatin analog SMS 201-995 (SMS) decreased the growth-promoting effect of bFGF. The maximal effect was observed at I nM SMS and the half-maximal effect at 20 pM SMS. Characterization of bFGF receptor-binding properties with [125I]bFGF revealed that AR4-2J cells exhibited 2 classes of bFGF binding site with respective KD values of 47 pM and 3 nM and binding capacities of 14 fmol and 0.9 pmol/10(6) cells. High-affinity receptors correlated with bFGF stimulation of AR4-2J cell growth, suggesting that the effects of bFGF are receptor-mediated.


Human c-erbB-2 proto-oncogene product as a target for bispecific-antibody-directed adoptive tumor immunotherapy.

To develop an efficient strategy for the targeting of anti-tumor effector cells, we prepared bispecific antibody (BsAb) containing anti-CD3 and an anti-c-erbB-2 proto-oncogene product. The prepared BsAb specifically reacts with both c-erbB-2-positive tumor cells and CD3+ CTL. Human CD4+ helper/killer T cells, induced from peripheral-blood mononuclear cells by activation with immobilized anti-CD3 monoclonal antibody (MAb) plus IL-2, showed no significant cytotoxicity against tumor cells. However, treatment of human CD4+ helper/killer cells with the BsAb caused the induction of specific cytotoxicity against c-erbB-2-positive tumor cells. CD4+ helper/killer cells also produced significant amounts of IL-2 during co-culture with c-erbB-2-positive tumor cells in the presence of the BsAb. Moreover, by combination with the BsAb, CD4+ helper/killer cells showed a strong in vivo anti-tumor effect against c-erbB-2 transfectant or human colon-cancer cells implanted in nude mice. Our results strongly suggest that the c-erbB-2 proto-oncogene product on human tumor cells may be a good target for BsAb-directed adoptive tumor immunotherapy.


Clinical experience with a new norgestimate-containing oral contraceptive.

Research in the area of oral contraception currently focuses on the development of selective progestogens that combine targeted progestational and antiovulatory activity with a minimal potential for androgenicity. The present dual-center study was conducted to investigate the efficacy, tolerability, and safety of a new monophasic oral contraceptive (OC) containing 250 micrograms norgestimate in combination with 35 micrograms ethinyl estradiol (Ortho-Cyclen or Cilest). Ninety-seven healthy women of childbearing age participated in the study: 37 received the new norgestimate/ethinyl estradiol combination OC as primary therapy and 31 were switched over from other OCs. The norgestimate/ethinyl estradiol formulation was well tolerated and was associated with excellent cycle control. After six cycles of use, there were no statistically significant differences in the incidence of spotting or breakthrough bleeding compared with baseline, nor were there any significant changes in the incidence of headache, nausea, or mastalgia. Body weights remained constant for the duration of the study, as did systolic and diastolic blood pressures. Of particular note was the absence of any statistically significant alterations in metabolic parameters, including blood glucose or lipoprotein levels. These findings are consistent with the results of several other European studies and indicate that the norgestimate/ethinyl estradiol combination OC combines superior cycle control with minimal risk of androgenic side effects.


Oral contraception: past, present, and future perspectives.

Oral contraceptives (OCs) were initially approved for unrestricted use in 1960 in the United States and have been used and studied extensively for 30 years. The initial formulations contained a fixed dose of estrogen and progestogen ingested for 21 days, with a seven-day pill-free interval. Subsequent formulations contained a sequential estrogen dose, a progestogen alone given daily, and variable doses of both progestogen and estrogen. Although the estrogen and progestogen doses employed in currently marketed OCs are markedly lower than those used in the OCs of the 1960s and 1970s, the excellent contraceptive efficacy of these compounds has not been compromised. The estrogen component produces a dose-related increase in serum globulin concentrations, triglycerides, and high-density lipoprotein (HDL) cholesterol, along with a decrease in low-density lipoprotein (LDL) cholesterol, while the progestogen component causes peripheral insulin resistance, a decrease in HDL cholesterol, an increase in LDL cholesterol, and various androgenic effects. The effect of nicotine on thromboxane release acts synergistically with the elevated serum clotting factors to increase the incidence of both arterial and venous thrombotic events, particularly in women smokers over 35 years of age. However, there is no evidence of increased risk of myocardial infarction or stroke in healthy, nonsmoking women of any age who use OCs containing less than 50 micrograms estrogen. Likewise, the lower-dose estrogen/progestogen formulations do not have a clinically significant effect on glucose metabolism and have a neutral effect on lipoprotein metabolism. In addition, the many noncontraceptive health benefits associated with OCs are maintained with the lower-dose formulations. Thus, the low-dose formulations should improve the overall health of healthy, nonsmoking women as well as effectively prevent unwanted pregnancy.


Differential expression of human corneal and perilimbal ICAM-1 by inflammatory cytokines.

The mechanisms that regulate corneal infiltration by circulating leukocytes in inflammatory diseases are poorly understood. In this study, we investigated the effects of pro-inflammatory cytokines on corneal endothelial (CE) and stromal (CS) expression of intercellular adhesion molecule-1 (ICAM-1), a specialized cell surface glycoprotein that binds the leukocyte function antigen-1 (LFA-1) receptor present on all leukocytes and enhances immune responses. Using specific monoclonal antibody (mAb) to ICAM-1, immunohistochemical staining of intact human corneas resulted in discrete, granular reaction product in CE and CS cells as well as perilimbal vascular endothelium that increased dramatically when exposed to human recombinant interleukin-1-beta (rIL-1 beta), tumor necrosis factor-alpha (rTNF-alpha), and interferon-gamma (rIFN-gamma). Immunoreactive ICAM-1 in CE and CS cells was differentially increased by each of these cytokines. In contrast, immunoreactive endothelial-leukocyte adhesion molecule-1 and vascular cell adhesion molecule-1 were not detected in any CE or CS cells of unstimulated or cytokine-stimulated corneas. In standardized leukocyte adherence assays, neutrophil binding to CE surfaces of whole corneas increased significantly upon exposure to rIL-1 beta, rTNF-alpha, or rIFN-gamma (P less than 0.001). In parallel assays, mAb to ICAM-1 on CE cells or subunits of LFA-1 receptors on leukocytes, but not control mAb, significantly blocked leukocyte binding to unstimulated (P less than 0.01) or rIFN-gamma-stimulated corneas (P less than 0.001). Our results indicate that: (1) ICAM-1 is expressed at low levels on unstimulated CE cells, CS cells, and perilimbal vascular endothelium; (2) ICAM-1 may be augmented differentially in corneal and perilimbal tissue by pro-inflammatory cytokines; and (3) ICAM-1 is a functional ligand mediating corneal-leukocyte binding. Differential expression of ICAM-1 within corneal tissue may regulate keratitic precipitate formation, leukocyte trafficking and accumulation, and localized generation of immune responses.


Insertion/deletion polymorphism and other restriction fragment length polymorphisms in the MCC gene.

The MCC gene is a candidate as a tumor suppressor gene for colorectal neoplasms. Further, MCC is tightly linked to the familial adenomatous polyposis (FAP) locus by linkage and physical analysis. Hence, restriction fragment length polymorphisms (RFLPs) of this gene might be very useful for presymptomatic diagnosis of individuals in families segregating mutant alleles of the APC gene. Here we report the identification of five polymorphic systems in MCC gene (both cDNA and genomic), one of which is an insertion/deletion polymorphism that is detectable by a polymerase chain reaction method. These five RFLP systems should be useful for linkage studies in FAP and for examining loss of heterozygosity at this locus in colonic polyps and tumors.


Beta 2 agonist therapy: oral versus inhaled delivery.

Although there is a place for administering oral medication, the inhaled route of administration for both bronchodilator and prophylactic purposes has been definitely shown to be the preferred route in numerous studies. The rapid onset of action, equivalent activity, and reduction in systemic effects all should lead the clinician to this form of therapy. Care must be taken to ensure proper utilization of the different types of inhalational medication to optimize their effectiveness.


Expression and characterization of kinase-active v-erbB protein using a baculovirus vector system.

The v-erbB gene is an oncogene of the avian erythroblastosis virus encoding a protein that is a truncated version of the epidermal growth factor receptor. The v-erbB protein was expressed alone or as polyhedrin-erbB fusion proteins using the Bombyx mori nuclear polyhedrosis virus vector. The expression level of the fusion protein whose polyhedrin portion consisted of only 8 amino-terminal amino acids was more than ten times higher than that of the non-fusion protein. Studies with tunicamycin showed that the recombinant v-erbB proteins were glycosylated. The recombinant protein autophosphorylated tyrosine residues, and phosphorylated a synthetic tyrosine-containing peptide and lipocortin I. These observations indicate that functional v-erbB protein can be expressed in silkworm-derived cells, and furthermore, that this system can be used for large-scale production.


Isolation and characterization of a Xenopus cDNA which encodes a homeodomain highly homologous to Drosophila Distal-less.

A novel homeobox gene of Xenopus was isolated from the ovary cDNA library. The homeodomain of the encoded protein was homologous to that of Drosophila Distal-less (Dll), and the gene was termed Xdll. The mRNA exists in a large amount in ovary, and in a small amount in testis, but was not detected in muscle, kidney, gut, and liver. The mRNA also occurs in a large amount in oocytes and is maintained in unfertilized eggs and cleavage stage embryos as a maternal mRNA at a low but distinctly detectable level. The amount of the mRNA per embryo increases gradually in later stages by zygotic expression. Embryo dissection experiment revealed that the transcript is abundant in the anterior region at the neurula stage, suggesting that Xdll may play a role in the establishment of the structures in the anterior part of the embryo.


Characterization of two site-specifically mutated human dihydrolipoamide dehydrogenases (His-452----Gln and Glu-457----Gln).

Two site-specifically mutated human dihydrolipoamide dehydrogenases (His-452----Gln and Glu-457----Gln) were expressed in pyruvate dehydrogenase complex-deletion mutant Escherichia coli JRG1342. The expressed mutant E3s were purified to near homogeneity using DEAE-Sephacel and hydroxyapatite columns. The initial velocity measurements in the absence of products for the Gln-452 mutant E3 in the direction of NAD+ reduction showed parallel lines in double-reciprocal plots, indicating that the mutant E3, like wild-type enzyme, catalyzed E3 reaction via a ping-pong mechanism. The specific activity of the Gln-452 mutant E3 was about 0.2% of that of wild-type enzyme. Its Km for dihydrolipoamide was dramatically increased by 63-fold. The substitution of His-452 to Gln resulted in a destabilization of the transition state of human E3 catalysis by about 6.4 kcal mol-1. The Gln-457 mutant E3, unlike wild-type enzyme, catalyzed E3 reaction via a sequential mechanism in the direction of NAD+ reduction based on the intersecting lines shown on double-reciprocal plots. Its specific activity decreased to 28% of that of wild-type enzyme. Its Km for dihydrolipoamide increased about 4.3-fold. The substitution of Glu-457 to Gln resulted in a destabilization of the transition state by about 1.7 kcal mol-1. These results indicate that His-452, which is a possible proton acceptor/donor in human E3 reaction, is critical to human E3 catalysis and that the local environment around His-452 and Glu-457, which are suggested to be hydrogen-bonded, is important in the binding of dihydrolipoamide to the enzyme.


Identification and biochemical characterization of novel putative substrates for the epidermal growth factor receptor kinase.

To gain insight into the mechanisms which control the mitogenic response to epidermal growth factor (EGF), we have partially purified and characterized several intracellular proteins which are phosphorylated on tyrosine residues following activation of the epidermal growth factor receptor (EGFR). Partial purification was achieved by immunoaffinity chromatography using immobilized anti-phosphotyrosine antibodies. Antisera generated against the partially purified proteins were used to identify at least five novel EGFR putative substrates, designated, on the basis of their apparent molecular weight, p97, p68, p61, p56, and p23. All of these proteins became specifically phosphorylated on tyrosine after EGF treatment of intact cells, as assessed by phosphoamino acid analysis, and none of them represented an EGFR degradation product. The phosphorylation of these proteins appeared to be relatively specific for the EGFR. In particular, an EGFR-related kinase, erbB-2 was much less efficient than EGFR at phosphorylating p97, p56, and p23 and incapable of phosphorylating p68. The identification of these novel EGFR putative substrates should lead to a better understanding of the mechanisms controlling the specificity of EGFR-mediated mitogenic signaling.


Cloning and characterization of the P subunit of glycine decarboxylase from pea (Pisum sativum).

A pea leaf cDNA library constructed in lambda gt11 was screened with an antibody raised to the P subunit of glycine decarboxylase. One of the positive clones isolated was sequenced and shown to contain an open reading frame, which encoded the entire P subunit polypeptide. Aligning the deduced amino acid sequence with the amino acid sequence determined directly from the NH2 terminus of the mature P subunit shows the presence of a putative 86 amino acid leader sequence, presumably required for import into the mitochondria, and gives a Mr of the mature protein of 105,000. Comparison of this deduced amino acid sequence with the sequence of a pyridoxal phosphate-containing peptide isolated from the P subunit of chicken liver glycine decarboxylase shows remarkable conservation. The P subunit, however, shows little sequence homology with other published amino acid decarboxylases. Expression of the P subunit mRNA shows a pattern very similar to that of the corresponding polypeptide: it is strongly light induced and is expressed at a much higher level in leaves than in other tissues. Southern blot analysis suggests that the P subunit is encoded by a small multigene family.


Isolation and characterization of the acetyl-CoA synthetase from Penicillium chrysogenum. Involvement of this enzyme in the biosynthesis of penicillins.

Acetyl-CoA synthetase (ACS) of Penicillium chrysogenum was purified to homogeneity (745-fold) from fungal cultures grown in a chemically defined medium containing acetate as the main carbon source. The enzyme showed maximal rate of catalysis when incubated in 50 mM HCl-Tris buffer, pH 8.0, at 37 degrees C. Under these conditions, ACS showed hyperbolic behavior against acetate, CoA, and ATP; the Km values calculated for these substrates were 6.8, 0.18, and 17 mM, respectively. ACS recognized as substrates not only acetate but also several fatty acids ranging between C2 and C8 and some aromatic molecules (phenylacetic, 2-thiopheneacetic, and 3-thiopheneacetic acids). ATP can be replaced by ADP although, in this case, a lower activity was observed (37%). ACS in inhibited by some thiol reagents (5,5'-dithiobis(nitrobenzoic acid), N-ethylmaleimide, p-chloromercuribenzoate) and divalent cations (Zn2+, Cu2+, and Hg2+), whereas it was stimulated when the reaction mixtures contained 1 mM dithiothreitol, reduced glutathione, or 2-mercaptoethanol. The calculated molecular mass of ACS was 139 +/- 1 kDa, and the native enzyme is composed of two apparent identical subunits (70 kDa) in an alpha 2 oligomeric structure. ACS activity was regulated "in vivo" by carbon catabolite inactivation when glucose was taken up by cells in which the enzyme had been previously induced. This enzyme can be coupled "in vitro" to acyl-CoA:6-aminopenicillanic acid acyltransferase from P. chrysogenum, thus allowing the reconstitution of the functional enzymatic system which catalyzes the two latter reactions responsible for the biosynthesis of different penicillins. The ACS from Aspergillus nidulans can also be coupled to 6-aminopenicillanic acid acyltransferase to synthesize penicillins. These results strongly indicate that this enzyme can catalyze the activation (to their CoA thioesters) of some of the side-chain precursors required in these two fungi for the production of several penicillins. All these data are reported here for the first time.


Identification of two molecular defects in a child with leukocyte adherence deficiency.

Children with leukocyte adherence deficiency (LAD), or leukocyte cell adhesion molecule deficiency, experience recurrent, life-threatening bacterial infections related to severe deficiency in surface expression of the leukocyte integrin molecules. The leukocyte integrins consist of a common CD18 (beta) subunit and individual, noncovalently associated alpha subunits designated CD11a, CD11b, and CD11c. Defects in the CD18 subunit prevent surface expression of the CD11/CD18 complexes in children with this disease. We investigated the molecular basis of the disease in a child with the severe deficiency form of LAD and identified two molecular defects in the CD18 subunit. The first defect is a single-base pair C----T transposition resulting in an amino acid substitution of a leucine for a proline at amino acid 178. This amino acid substitution is located in a region that is highly conserved among the integrin beta subunits and where two previous defects have been located in LAD. The second mutation involves a deletion of 220 base pairs in the cDNA coding for a portion of the extracellular domain and results in a frameshift into a premature stop codon. The deleted region corresponds to a single exon in the CD18 gene. Identification of these two molecular defects in a single child with this disease indicates the compound heterozygous nature of the disorder in this child and identifies regions of the CD18 subunit that may be important for CD11/CD18 heterodimer formation and surface expression.


High-carbohydrate, low-fat diet? Negative.

Triglyceride levels rise with increased carbohydrate intake, according to a long-term study of normal children from several countries. Moreover, carbohydrate raises blood glucose levels and insulin requirements.


Is tight blood sugar control effective and justified? Affirmative.

Continuous subcutaneous insulin infusion can delay progression of both retinopathy and nephropathy. In fact, very few insulin-dependent patients developed clinical renal disease while on this treatment.


Tight glycemic control? Negative.

Many poorly controlled patients do not develop proliferative retinopathy and nephropathy, whereas some tightly controlled patients do. In any event, normalization of blood glucose may be impossible and dangerous.


Are oral hypoglycemic agents likely to benefit NIDDM patients? Affirmative.

The sulfonylureas may prevent progression from impaired glucose tolerance to diabetes as well as control hyperglycemia. Patient response depends on the physiologic status of insulin secretion and effectiveness, however.


Oral hypoglycemic agents? Negative.

I am convinced that oral hypoglycemic therapy is associated with increased cardiovascular mortality. But the more important issue is whether the benefits of these agents are worth the risk.


'Syndrome X': is it a significant cause of hypertension? Affirmative.

A number of biologic actions of insulin lend credence to the hypothesis that insulin resistance can produce hypertension, perhaps by stimulating a hyperkinetic circulation. But it may do so only in lean individuals.


Glycosylation of proteins and microangiopathy.

Diabetic complications may result from chronic glycosylation of protein within cells and in the extracellular matrix. Prevention of glycosylation with aminoguanidine has forestalled complications in experimental diabetes.


Proteinuria and microalbuminuria as predictors of nephropathy.

Measurement of proteinuria, microalbuminuria, and sodium-lithium countertransport in red cells has no practical value. A low-protein diet and ACE inhibitor therapy are currently the best way to retard progression of diabetic renal disease.


Lipoprotein abnormalities in diabetes mellitus.

In most diabetic patients, insulin or sulfonylurea treatment also resolves the characteristic triglyceride elevation. However, patients with central obesity or familial hypertriglyceridemia may require a lipid-lowering drug.


Should NIDDM patients be on high-carbohydrate, low-fat diets? Affirmative.

Most diet studies are too short, capturing only the temporary rise in triglycerides after a high-carbohydrate, low-fat diet is begun. Over time, triglycerides and total cholesterol will fall while HDL cholesterol is maintained.


Psychotic (delusional) depression: a meta-analysis of physical treatments.

Literature reviews have suggested that combination antidepressant/antipsychotic drug therapy and electroconvulsive therapy (ECT) are of comparable efficacy in treating psychotic depression, and distinctly superior to antidepressant alone or antipsychotic alone. We undertook a meta-analysis of 44 studies, and focussed on those three principal treatment options. There was a trend for ECT to be superior to combination drug therapy, with bilateral ECT being suggested as distinctly more effective than unilateral, and ECT was demonstrated to be significantly superior to tricyclic drug alone. Combination drug therapy ranked as more effective than antipsychotic alone and than antidepressant alone, but that greater efficacy was not significant.


The effect of a selective alpha 2-adrenergic receptor antagonist (midaglizole) on the cyclic 3',5'-adenosine monophosphate production in human mononuclear cells.

A selective alpha 2-adrenergic antagonist, midaglizole, has been recently reported to have efficacy in patients with asthma. To understand the mechanisms, we investigated the effect of midaglizole on the cyclic 3', 5'-adenosine monophosphate (cAMP) production in human mononuclear cells (MNCs). MNCs were separated by a histopaque gradient from 10 normal subjects and 10 subjects with asthma. cAMP was measured by RIA kits. Midaglizole (10 mumol/L) significantly enhanced isoproterenol-induced cAMP production in both groups, although midaglizole (from 1 to 100 mumol/L) did not increase the cAMP production by itself. The percent increase in cAMP was more in subjects with asthma (183.8%) than in normal subjects (140.4%); however, the absolute increase was not different. Platelet-activating factor has been demonstrated to inhibit beta-agonist-induced cAMP production in several mammalian tissues, including human MNCs. Midaglizole also prevented platelet-activating factor-induced desensitization of the cAMP response to isoproterenol in MNCs. These findings suggest that midaglizole may be a useful additional agent for the therapy of bronchial asthma through an enhancement of the cAMP production.


Stimulation of human naive and memory T helper cells with bacterial superantigen. Naive CD4+45RA+ T cells require a costimulatory signal mediated through the LFA-1/ICAM-1 pathway.

The role of the accessory molecule ICAM-1 in activation of subpopulations of human T cells was examined using the bacterial superantigen staphylococcal enterotoxin A (SEA) as a MHC class II and TCR-dependent polyclonal T cell activator. Human T cells responded with different sensitivity to SEA when presented on mouse accessory cells expressing a human transfected MHC class II gene product. Mouse L cells cotransfected with both MHC class II (DR2A or DR7) and ICAM-1-stimulated T cells at 100-fold lower concentrations of SEA as compared to the single transfected cells. mAb reacting with the CD11a, CD18, or ICAM-1 molecules efficiently inhibited T cell activation with the cotransfected HLA-DR2A/ICAM-1 cell but did not influence T cell activation with the HLA-DR2A single transfected cell. Analysis of the ICAM-1 requirement on CD4+ memory (CD4+45RO+) and naive (CD4+45RA+) T cells revealed that CD4+45RA+ naive Th cells were hyporesponsive to SEA-induced activation with the HLA-DR2A single transfectant. However, cotransfection of ICAM-1 enabled these cells to respond to low doses of SEA implicating that they are more dependent on accessory molecules than the CD4+45RO+ cells. rICAM-1 immobilized on a plastic surface, was able to strongly costimulate SEA-induced T cell activation with the HLA-DR2A single transfectant, suggesting that costimulatory signals mediated to the T cells through LFA-1 can be delivered physically separated from the TCR signal. CD4+45RO+ memory and CD4+45RA+ naive Th cells apparently differ in their capacities to be activated by SEA bound to HLA-DR. Although the TCR molecule densities are similar in these two subsets, costimulation with ICAM-1 is required for activation of the CD4+45RA+, but not the CD4+45RO+ T cell subset at 1 to 10,000 ng/ml concentrations of SEA. This observation indicates different activation thresholds of naive and memory Th cells when triggering the TCR over a wide dose interval of superantigen.