Different structural constraints for recognition of mouse H-2Kd and -Kk antigens by alloimmune cytolytic T lymphocytes.

We have constructed a new series of hybrid genes among the H-2Kd,-Kk, and -Kb. The site of recombination occurs in the third exon, encoding the alpha 2 domain, and divides this domain into two parts, alpha 2A and alpha 2B. The novel genes differ only in the COOH-terminal half of the alpha 2 domain, i.e., the alpha 2B region. This region, comprising residues 142-182, contains a limited number of amino acid differences between the three alleles. The hybrid genes have been introduced into 1T 22-6 cells (H-2q), and cell surface expression of hybrid antigens was verified. Cells expressing different types of hybrid antigens have been examined for their susceptibility to lysis by cytotoxic T lymphocytes directed either against the H-2Kd antigen or the H-2Kk antigen. Our results show that the alpha 1 and alpha 2A domains of the H-2Kk antigen can constitute target molecules for alloimmune anti-Kk T cells, whereas the alpha 2B region, when exchanged for Kd or Kb sequences, plays only a limited role. In contrast, the alpha 1 and alpha 2A domains of Kd are not sufficient to be recognized by alloimmune anti-Kd T cells. In this instance, the alpha 2B domain seems to play an essential role. This region has undergone several amino acid substitutions involving charged residues.

Mitogenome of the blood feeding leech Haementeria acuecueyetzin (Hirudinida: Glossiphoniidae) from Tabasco, Mexico.

Here, we present the mitogenome of the blood feeding leech Haementeria acuecueyetzin (Hirudinida: Glossiphoniidae) based on specimens collected in Tabasco, Mexico. The circular genome is 14,985 bp in length, and consists of 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and an AT-rich control region. Phylogenetic analysis based on the 13 protein-coding genes and two rRNA genes places H. acuecueyetzin sister to H. officinalis within the family Glossiphoniidae. Mitochondrial gene order in H. acuecueyetzin is consistent with other members of Clitellata with no evidence of gene gain/loss, duplication, or rearrangement.

The endoplasmic reticulum retention signal of the E3/19K protein of adenovirus type 2 consists of three separate amino acid segments at the carboxy terminus.

The E3/19K protein of adenovirus type 2 is a resident of the ER. Immediately after synthesis it binds to human major histocompatibility complex class I antigens and prevents their departure from the ER compartment. The ER retention signal of the E3/19K protein is contained within the 15 amino acids that protrude on the cytoplasmic side at the carboxy terminus of the protein. To define the ER retention sequence in more detail, we have generated 10 mutants of the E3/19K protein that differ only within this segment. Analysis of the rate of intracellular transport and cell surface expression of HLA antigens associated to these mutants, show that the sequences Ser-Phe-Ile, located in the middle of the 15-residue segment and Met-Pro, at the extreme carboxy terminus, are crucial for retention. Four charged residues, Asp-Glu-Lys-Lys, are located between these two retention elements but are of little or no importance. The basic cluster of amino acids close to the membrane also has some effect on retention. Thus, the retention signal of the E3/19K protein is not a contiguous sequence of amino acids but has a complex spatial arrangement.

Translocation of peptides through microsomal membranes is a rapid process and promotes assembly of HLA-B27 heavy chain and beta 2-microglobulin translated in vitro.

We have translated major histocompatibility complex (MHC) class I heavy chains and human beta 2-microglobulin in vitro in the presence of microsomal membranes and a peptide from the nucleoprotein of influenza A. This peptide stimulates assembly of HLA-B27 heavy chain and beta 2-microglobulin about fivefold. By modifying this peptide to contain biotin at its amino terminus, we could precipitate HLA-B27 heavy chains with immobilized streptavidin, thereby directly demonstrating class I heavy chain-peptide association under close to physiological conditions. The biotin-modified peptide stimulates assembly to the same extent as the unmodified peptide. Both peptides bind to the same site on the HLA-B27 molecule. Immediately after synthesis of the HLA-B27 heavy chain has been completed, it assembles with beta 2-microglobulin and peptide. These interactions occur in the lumen of the microsomes (endoplasmic reticulum), demonstrating that the peptide must cross the microsomal membrane in order to promote assembly. The transfer of peptide across the microsomal membrane is a rapid process, as peptide binding to heavy chain-beta 2-microglobulin complexes is observed in less than 1 min after addition of peptide. By using microsomes deficient of beta 2-microglobulin (from Daudi cells), we find a strict requirement of beta 2-microglobulin for detection of peptide interaction with the MHC class I heavy chain. Furthermore, we show that heavy chain interaction with beta 2-microglobulin is likely to precede peptide binding. Biotin-modified peptides are likely to become a valuable tool in studying MHC antigen interaction and assembly.

Prepartum and postpartum open-field behavior and maternal responsiveness in mice bidirectionally selected for open-field thigmotaxis.

The authors examined pre- and postpartum open-field (OF) behavior and maternal responsiveness in mice that they bidirectionally selected for OF thigmotaxis. The authors tested 40 female mice under 3 conditions: prepartum OF, postpartum OF, and a pup retrieval test. In both OF conditions, the high OF thigmotaxis (HOFT) mice were more thigmotactic but explored and reared less than the low OF thigmotaxis (LOFT) mice, indicating that the HOFT mice were more emotional. In the postpartum condition, the HOFT mothers also defecated more and ambulated less than the LOFT mothers. The increase in grooming after parturition was more conspicuous among the LOFT mothers than among the HOFT mothers. The LOFT mothers were also more attracted to their pups in the OF, but the retrieval test did not show any substantial line differences. The results suggested that the line difference in emotionality was more pronounced during lactation than during pregnancy, although parturition exerted no effect on thigmotaxis.

Twenty-three generations of mice bidirectionally selected for open-field thigmotaxis: selection response and repeated exposure to the open field.

We examined: (a) the response to bidirectional selection for open-field (OF) thigmotaxis in mice for 23 generations and (b) the effects of repeated exposure (during 5 days) on different OF behaviors in the selectively bred high OF thigmotaxis (HOFT) and low OF thigmotaxis (LOFT) mice. A total of 2049 mice were used in the study. Prior to the testing in the selection experiment, the mice were exposed to the OF apparatus for approximately 2 min on each of 4 consecutive days. Thus, the selection was based on the scores registered on the 5th day after the four habituation periods. The HOFT mice were more thigmotactic than the LOFT mice in almost each generation. The HOFT mice also tended to rear less than the LOFT mice, which was explained by the inverse relationship between emotionality and exploratory tendencies. The lines did not generally differ in ambulation. Sex differences were found in thigmotaxis, ambulation, and rearing. In the repeated exposure experiment, the development of nine different OF behaviors across the 5 days of testing was addressed. Both lines ambulated, explored, and reared most on the 1st, 4th, and 5th days. Grooming and radial latency decreased and thigmotaxis increased linearly across the testing days. Line differences were found in ambulation, exploration, grooming, and rearing, while sex differences were manifested in ambulation and exploration. The line difference in thigmotaxis was evident only on the 5th day. Temporal changes were partially at variance with the general assumptions. OF thigmotaxis was found to be a powerful characteristic for producing two diverging lines of mice.

From the Worm in a Bottle of Mezcal: iDNA Confirmation of a Leech Parasitizing the Antillean Manatee.

Invertebrate-derived ingested DNA (iDNA) is quickly proving to be a valuable, non-invasive tool for monitoring vertebrate species of conservation concern. Using the DNA barcoding locus, we successfully identified both the blood-feeding leech Haementeria acuecueyetzin and its blood meal-the latter is shown to be derived from the Caribbean manatee, Trichechus manatus . DNA amplification was successful despite the fact that the specimen was fixed in Mezcal (a beverage distilled from agave). We report the first confirmed case of a leech feeding on a manatee, the first record of H. acuecueyetzin for the State of Chiapas and, to our knowledge, the first case of successful DNA amplification of a biological sample fixed in Mezcal other than the caterpillar "worms" more commonly found in that beverage.

Depression, anxiety, hostility and hysterectomy.

Sixty-five women (aged 32 - 54 yrs) were assessed at 2 months before to 8 months after total abdominal hysterectomy on four separate occasions. Beck's Depression Inventory (BDI), Taylor's Manifest Anxiety Scale (TMAS), the Buss-Durkee Hostility Inventory (BDHI), Measurement of Masculinity-Femininity (MF), Likert scales and semantic differentials for psychological, somatic and sexual factors varied as assessment tools. High-dysphoric and low-dysphoric women were compared with regard to hysterectomy outcomes. Married nulliparae suffered from enhanced depression post-surgery. Pre-surgery anxiety, back pain and lack of dyspareunia contributed to post-surgery anxiety. Pre-surgery anxiety was related to life crises. Pre- and post-surgery hostility occurred in conjunction with poor sexual gratification. Post-hysterectomy health improved, but quality of sexual relationship was impaired. Partner support and knowledge counteracted hysterectomy aftermath. Post-hysterectomy symptoms constituted a continuum to pre-surgery signs of depression, anxiety or hostility.

Mice selectively bred for open-field thigmotaxis: life span and stability of the selection trait.

In 2 experiments, the authors examined 69 mice selectively bred for high or low levels of open-field (OF) thigmotactic behavior (high open-field thigmotaxis [HOFT] and low open-field thigmotaxis [LOFT], respectively). They found that the strains differed in defecation during the 60-min exposure to the OF. Furthermore, the strains differed with regard to their life spans: The more thigmotactic HOFT mice lived longer than the LOFT mice. The strains were not differentiated by food intake or excretion. The strain difference in thigmotaxis was not age dependent, and it persisted in the home-cage condition as well. Neither the location (center or wall) of the starting point nor the shape (circular or square) of the OF arena affected the difference in wall-seeking behavior between the two strains. The authors concluded that the difference in thigmotaxis (or emotionality) between the HOFT and LOFT mice is a stable and robust feature of these animals.

Reduced medication and normalization of vascular structure, but continued hypertension in renovascular patients after revascularization.

OBJECTIVE: The effect of invasive treatment of renal artery stenosis on the use of antihypertensive medication, blood pressure, and morphology and function of resistance arteries was investigated in 14 renovascular hypertensive patients before and after treatment compared to normotensive controls. METHODS: Use of antihypertensive medication was calculated as defined daily doses (DDD). Resistance arteries were taken from gluteal subcutaneous biopsies and analyzed in a myograph. RESULTS: Prior to invasive treatment, blood pressure of the patients was elevated compared to normotensive controls. Six months after technically successful invasive treatment, patients were still hypertensive compared to time-matched controls. The use of antihypertensive medication was reduced from 4.4+/-0.7 DDD before invasive treatment to 3.0+/-0.6 DDD 6 months after treatment. Vascular structure of resistance arteries, expressed as media/lumen ratio (media thickness to diameter), was greater in patients before invasive treatment (10.7+/-1.0%) compared to normotensive controls (7.9+/-0.8%). Media/lumen ratio of resistance arteries was reduced to that of the controls 6 months after invasive treatment despite the remaining hypertension. The functional studies showed no difference in response to acetylcholine, adenosine, noradrenaline or angiotensin II between patients and controls before or after treatment. CONCLUSIONS: This study shows that hypertension and increased media/lumen ratio of resistance arteries prevail in renovascular hypertensive patients despite antihypertensive medication and that invasive treatment is of benefit as regards use of antihypertensive medication. The study provides the novel information that the remaining hypertension is not due to uncorrected media/lumen ratio of the resistance arteries.

Studies of the renin-angiotensin system in the wall of rat femoral resistance vessels.

The responses to angiotensinogen, angiotensinogen-(1-14) (tetradecapeptide, TDP), angiotensin I, and angiotensin II and the effect of the renin inhibitor, CH-66, the angiotensin-converting enzyme (ACE) inhibitor, perindopril, and the receptor antagonist, saralasin, were investigated in isolated femoral resistance arteries of the rat. The response to angiotensinogen in the presence of kallikrein was also investigated. Angiotensin I and TDP elicited a contraction which was not reduced by CH-66 but was inhibited by perindopril and saralasin. The response to angiotensinogen was small and not blocked by saralasin but the response to angiotensinogen that was mixed with renin for a few seconds was saralasin-sensitive and perindopril and CH-66 showed a tendency to block this response. The response to angiotensinogen was enhanced in the presence of kallikrein. These results suggest (1) the presence of a partial renin-angiotensin system (RAS) in this preparation, (2) that TDP is not converted via renin while both angiotensin I and TDP are converted through ACE in this preparation and (3) that the responses to angiotensinogen and TDP are different.

Time course of action of amlodipine and felodipine in the rat is most rapid in small arteries.

The time course of action of amlodipine was compared to that of felodipine in rat mesenteric resistance arteries and aorta. Both amlodipine and felodipine caused a concentration-dependent relaxation of K(+)-depolarized resistance arteries: with amlodipine 3 x 10(-8) M and felodipine 10(-9) M, complete relaxation was reached after 40 min and 10 min, respectively. Furthermore, in resistance arteries, the time course of action of both drugs was shortest in vessels with the smallest diameter. In aorta, both drugs caused a marked relaxation of K(+)-induced tone, without reaching a maximal effect within 2 h. Recovery of K(+)-induced tone after both drugs was complete in resistance arteries, but not aorta, within 2 h. In resistance arteries exposed to K+ depolarization or noradrenaline, both drugs displayed the characteristics of 1,4-dihydropyridine Ca2+ channel antagonists. The results show that amlodipine was slower to have an effect than felodipine, but that both drugs acted fastest in the smallest arteries.

Perceptions of problematic events and quality of care among patients and parents after successful therapy of the child's malignant disease.

Fifty-three children (mean age 6.4 at diagnosis, 12.8 years at completion of questionnaires) identified as having acute lymphoblastic leukaemia (48) or non-Hodgkin lymphoma (5) during the 11-year period of 1976-1986 participated in the present study. Patients' and parents' perceptions of malignancy-related changes in significant relationships, of treatment-related problematic events, as well as of the quality of care provided during induction and maintenance-therapy were assessed using questionnaires. The results indicated mainly positive changes in significant relationships during the chemotherapy. However, concurrent stressful life events affected the relationships adversely. Alopecia (hair loss) was the most problematic disease-related event for the patient and the patient's anorexia for the parents. Patients' complaints were most often about the quality of care during induction, namely pain, fear and insufficient information. Parents' complaints were about the lack of continuity in the staff-patient relationship during maintenance-therapy.

Open-field parameters and maze learning in aggressive and nonaggressive male mice.

Significant differences were observed in thigmotaxis, ambulation, and latency to move (time to start ambulating) between highly aggressive (TA) and low aggressive (TNA) male mice. The former displayed more thigmotaxis, ambulated more, and had a shorter latency to move than the TNA animals. Also they voided a greater number of urinary spots and defecated less than TNA. Further they were superior to the TNA mice in maze-learning capacity. The tendency to enter inner partitions of the field as well as total ambulation increased after learning by TA mice. The training toward nonaggressiveness of TA mice suppressed aggressive responses, thigmotaxis, and the number of urinary spots but enhanced defecation. All measures returned to their initial levels after one month of rest. The attacking behaviour of TA animals increased both thigmotaxis and ambulation.

Sex-related coping responses in mice selectively bred for aggression.

Sex differences in strategies of coping with novel situations were studied in three strains of mice with regard to metabolism and open-field and maze activity as well as learning-induced adjustment. The 140 mice were selectively bred for high (Turku Aggressive [TA]) and low (Turku Nonaggressive [TNA]) levels of aggressiveness and originated from a Swiss albino stock normally distributed [N] for aggressiveness. The results indicated that TNA sex differences are more similar to those of the control N mice as compared to those of TA mice. In maze learning, however, the sex differences of TA mice are more in agreement with those of the N strain. Recordings of metabolism and open-field as well as maze activity were correlates of both gender and strain. Sex differences in learning-induced open-field coping behavior were unrelated to strain.

Aggression: the dominant psychological response in children with malignant disease.

During the 11-yr. period of 1976 to 1986 leukemia or lymphoma treatment at the Children's Hospital, University of Helsinki was electively discontinued for the children in 90 different families. Of the 53 (59%) patients (mean age 6.4 yr. at diagnosis and 12.8 yr. at completion of questionnaires) who agreed to participate in the present study, 48 had acute lymphoblastic leukemia and five nonHodgkin lymphoma. Patients' and parents' impressions of the patients' psychological reactions during patients' prior chemotherapy were evaluated on parental and self-ratings. Also, knowledge of and presumed causes of the malignancy were studied. Patients' reactions of aggression, depression, eating disorders, hypersensitivity, phobic anxiety, death anxiety, and night terror were examined using factor analysis. Aggression, in the form of irritation and anger, was displayed more often by girls than by boys. Patients of families suffering from stress were prone to exhibit aggression in the form of mood changes, irritation, and anger. Patients with disease-related knowledge, as opposed to those less well informed, were less depressed. Discrepancies between parents' and patients' thoughts about the origin of the malignancy were noted.

Crossfostering in mice selectively bred for high and low levels of open-field thigmotaxis.

The main purpose of this research was to investigate whether the difference in open-field (OF) thigmotaxis between mice selectively bred for high and low levels of wall-seeking behavior originated from genetic or acquired sources. Unfostered, infostered, and crossfostered mice were compared in two experiments in which the effects of strain, sex, and fostering on ambulation, defecation, exploration, grooming, latency to move, radial latency, rearing, thigmotaxis, and urination were studied. These experiments revealed that OF thigmotaxis was unaffected by the foster condition and thus genetically determined. The selected strains of mice also diverged repeatedly with regard to exploration and rearing. The findings are in line with the previously described existence of an inverse relationship between emotionality and exploration.

A nucleoprotein peptide of influenza A virus stimulates assembly of HLA-B27 class I heavy chains and beta 2-microglobulin translated in vitro.

Most cytotoxic T lymphocytes (CTL) recognize epitopes of foreign viral proteins in association with class I major histocompatibility complex (MHC) molecules. Viral proteins synthesized in the cytoplasm require intracellular fragmentation and exposure to the class I antigens for the development of CTL responses. Although indirect evidence for binding of peptides to class I antigens has accumulated, direct binding has only been shown recently. The formation of complexes between peptide and class I antigen may occur in the endoplasmic reticulum (ER) and peptides have been shown to induce assembly of the class I complex. We have translated the messenger RNAs encoding HLA-B27 (subtype 2705) and beta 2-microglobulin in a rabbit reticulocyte lysate supplemented with human microsomal membranes (to mimic ER membranes), in the absence and presence of a peptide derived from the nucleoprotein (residues 384-394) of influenza A virus. This peptide induces CTL activity against target cells expressing the HLA-B27 antigen. Here we report direct evidence that the nucleoprotein peptide promotes assembly of the HLA-B27 heavy chain and beta 2-microglobulin, and that this can occur in the ER immediately after synthesis of the two proteins.

Co-expression of the human HLA-B27 class I antigen and the E3/19K protein of adenovirus-2 in insect cells using a baculovirus vector.

We have expressed the human MHC class I HLA-B27 antigen, human beta 2-microglobulin, and the E3/19K protein of adenovirus-2 in Spodoptera frugiperda insect cells (Sf9) by using the Autographa californica nuclear polyhedrosis virus. All genes were inserted under the strong polyhedrin promoter in the vector pVL941. The proteins were expressed at high levels, ranging from 1 to 8 mg protein per 3 x 10(9) cells. Both a full-length and a truncated form of HLA-B27 were expressed. The latter was terminated at the border of the membrane-spanning segment at the extracellular side of the membrane. The HLA-B27 antigens and the E3/19K protein showed considerable heterogeneity with respect to glycosylation. Only a small fraction of HLA-B27 was assembled (less than 5%) with beta 2-microglobulin. Nevertheless, we were able to find the heavy chain at the cell surface, and by co-infection with the recombinant virus for beta 2-microglobulin we observed an increase in cell surface expression. The E3/19K protein of adenovirus-2 blocks cell surface expression of HLA class I antigens in human cells and has a similar effect in insect cells. In contrast to beta 2-microglobulin it assembles efficiently to the heavy HLA-B27 chain, both the full-length and truncated forms. The E3/19K protein does not stimulate assembly of HLA-B27-beta 2-microglobulin. Due to these problems of assembly and the heterogeneity of glycosylation we predict that it will be difficult to use HLA antigens produced in insect cells for X-ray crystallographic studies.