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This study examined the effect of aerobic exercise training on vagal and sympathetic influences on the modulations of heart rate and systolic blood pressure in response to an oral glucose load in obese individuals with and without type 2 diabetes mellitus (T2D). Beat-to-beat arterial pressure and continuous electrocardiogram were measured after a 12-hour overnight fast and in response to glucose ingestion (75 g dextrose) in obese subjects with (T2D group, n = 23) and without (OB group, n = 36) T2D before and after 16 weeks of aerobic exercise training at moderate intensity. Autonomic modulation was assessed using spectral analysis of systolic blood pressure variability (BPV), heart rate variability (HRV), and analysis of baroreflex sensitivity (BRS). Glucose ingestion significantly increased low-frequency (LF(SBP)), low-frequency HRV (LF(RRI)), and the ratio of low- to high-frequency components of HRV (LF(RRI)/HF(RRI)), and decreased the high-frequency power (HF(RRI)) (P < .05). Exercise training increased LF(RRI) and LF(RRI)/HF(RRI) responses, and reduced HF(RRI) and LF(SBP) to glucose ingestion in both groups (P < .05), but increased fasted BRS in the OB group only (P < .05); glucose intake had no effect on BRS (P > .05). In conclusion, a 16-week exercise training program improved cardiac autonomic modulation in response to an oral glucose load in obese adults, independently of diabetes status, and in the absence of remarkable changes in body weight, body composition, fitness level, and glycemic control., (Copyright 2010 Elsevier Inc. All rights reserved.)

Obese individuals with metabolic syndrome are predicted to have a 5 times greater risk of developing type 2 diabetes and a 3 times greater risk of myocardial infarction than those without metabolic syndrome. Many obese patients with metabolic risk factors have an accumulation of triglycerides within the skeletal muscle fibers, ie, intramyocellular lipids (IMCL). Individuals with metabolic syndrome have a greater risk of developing IMCL depots, and these stores are related to increased cardiovascular risk and type 2 diabetes. A few studies have shown, moreover, that aging seems to result in an increase in IMCL stores, although little is known about how exercise influences these stores. In healthy young individuals, IMCL depots have been shown to decrease in response to acute exercise, but very little is known about the effects of exercise training on IMCL depots in obese individuals. Understanding how IMCL depots can be altered with exercise (both resistance and aerobic exercise) in older and obese individuals seems to be critical in preventing disease.

Increases in abdominal fat have been reported with menopause, but the impact of menopause on abdominal fat distribution (visceral vs subcutaneous) is still unclear. The objective of the study was to determine if abdominal fat content (volume) or distribution is altered with menopause. Magnetic resonance imaging was used to quantify total abdominal, subcutaneous, and visceral fat in 8 healthy women, both in the premenopausal state and 8 years later in the postmenopausal state. Physical activity (PA) and blood lipids were also measured. Body weight and waist circumference did not change with menopause (pre- vs postmenopause: body weight, 63.2 +/- 3.1 vs 63.9 +/- 2.5 kg; waist circumference, 92.1 +/- 4.6 vs 93.4 +/- 3.7 cm); however, total abdominal fat, subcutaneous fat, and visceral fat all significantly (P < .05) increased with menopause (pre- vs postmenopause: total, 27 154 +/- 4268 vs 34 717 +/- 3272 cm(3); subcutaneous, 19 981 +/- 3203 vs 24 918 +/- 2521 cm(3); visceral, 7173 +/- 1611 vs 9798 +/- 1644 cm(3)). Although absolute adiposity changed with menopause, relative fat distribution was not significantly different after menopause (pre- vs postmenopause: subcutaneous, 73% +/- 3% vs 71% +/- 3%; visceral, 26% +/- 3% vs 28% +/- 3%). Lean mass, fat mass, and PA, along with total cholesterol and triglyceride levels, did not change with menopause. High-density lipoprotein and low-density lipoprotein both increased (P < .05), and the ratio of total cholesterol to high-density lipoprotein decreased (P < .05) with menopause. As measured longitudinally with magnetic resonance imaging, total abdominal fat content increased with menopause despite no change in PA, body weight, or waist circumference; however, menopause did not affect the relative abdominal fat distribution in these women.

Objective: To compare autonomic function, measured during handgrip (HG) and cold pressor (CP), between obese with and without type 2 diabetes and non-obese women in fasting and post-glucose load states., Methods: Twelve obese women with type 2 diabetes (50 +/- 1 years), 15 obese women without type 2 diabetes (48 +/- 2 years), and 12 non-obese women (49 +/- 2 years) participated in this study. Heart rate variability (HRV) was determined during autonomic function tests, conducted in both the fasting state and after a glucose challenge (oral glucose tolerance test-OGTT)., Results: Obese women with and without diabetes and non-obese women responded similarly fasted and post-glucose challenge, such that in the fasted state low frequency power normalized (LF(nu)) to total power (TP), log transformed (Ln) low frequency to high frequency ratio (LnLF/HF) and heart rate (HR) significantly increased with the autonomic functional tasks (P < 0.05), whereas HF(nu) significantly decreased with the tasks (P < 0.05). Handgrip elicited a lower LnTP and a higher HR (P < 0.05) when compared to CP in the fasted state. In the glucose challenged state LF(nu), LnLF/HF and HR increased (P < 0.05) and HF(nu) significantly decreased (P < 0.05)., Interpretation: Results of autonomic testing did not differ between obese women, with and without diabetes, and non-obese women. The HG test elicited a greater reduction in HRV total power compared to the CP. This suggests that HG may be more useful when examining autonomic function in women with complicated diabetes.

This study examined the effect of glucose ingestion on cardiac autonomic function in nonobese women and obese women with and without type 2 diabetes mellitus. Heart rate variability was measured via continuous electrocardiogram, and beat-by-beat blood pressure was recorded using finger photoplethysmography (Portapres, TNO Biomedical Instrumentation, Amsterdam, The Netherlands) in a fasted state and in response to a 75-g glucose load in 42 middle-aged women (40-60 years). Upright tilt was also used as an orthostatic stress to provide a clinically relevant challenge to the cardiovascular system. Significant main effects for log-transformed (Ln) total power (TP, square milliseconds) were observed with upright tilt (P < .01) and glucose challenge (P < .05). LnTP decreased in all groups in both the fasted and fed state with upright tilt (P < .01), but glucose ingestion resulted in higher LnTP in the supine position only (P = .008). Tilt resulted in a significant main effect for low-frequency (LFnu, calculated in normalized units) and high-frequency (HFnu, calculated in normalized units) power (P < .000), whereas the glucose challenge had no effect on LFnu or HFnu power. LFnu approached significance for group differences (P = .07), such that the nonobese had lower LF power than either of the obese groups. Sympathovagal balance (LnLF/HF ratio) was affected by position (P < .000) and group (P < .05), with a lower LnLF/HF in the nonobese than in the obese women. Baroreceptor sensitivity decreased (P < .01) during upright tilt but was not changed by the glucose challenge. In conclusion, basal sympathovagal balance is higher in obese individuals with and without type 2 diabetes mellitus. Women with type 2 diabetes mellitus showed no differences in autonomic function with an orthostatic challenge or glucose load than nondiabetic, obese women. The glucose load did alter total spectral power in all of these middle-aged women but had no impact on baroreceptor sensitivity.

The Surgeon General and Centers for Disease Control and Prevention have recommended that multiple bouts of exercise can be accumulated throughout the day in lieu of the more traditional single, longer bout of exercise. Yet, conclusive evidence does not exist suggesting these 2 training modes provide similar health-related benefits on metabolic control, especially in individuals with type 2 diabetes. The purpose of this study was to determine if differences exist in glucose control when preceded by one 30-minute or three 10-minute bouts of exercise in women with type 2 diabetes. Nine individuals with type 2 diabetes (53 +/- 6 years old) and 6 control women (49 +/- 4 years old) completed 3 randomly ordered oral glucose tolerance tests (OGTTs). Two of the OGTTs were preceded the day prior by moderate exercise (approximately 60% of Vo2peak), either one 30-minute or three 10-minute bouts, whereas the third OGTT was used as a control day with no exercise performed 3 days prior. Glucose and insulin were measured every 30 minutes for 4 hours during the OGTT. Individuals with type 2 diabetes exhibited a greater overall glucose response than the controls (P < .05), but the glucose response to the OGTT was not different between the 3 conditions within each group (2-hour glucose: multiple bout, 14.3 +/- 3.2 vs 5.0 +/- 1.7; single bout, 14.1 +/- 3.0 vs 4.7 +/- 1.5; control day, 14.6 +/- 2.7 vs 4.9 +/- 4.9 mmol/L). Glucose area under the curve analysis resulted in similar findings. As expected, the group with type 2 diabetes had greater fasting insulin levels compared with the control group for all exercise conditions (multiple bout: 4.5 +/- 1.2 vs 0.3 +/- 0.2; single bout: 6.4 +/- 1.6 vs 0.9 +/- 0.4; control day: 5.7 +/- 1.8 vs 1.5 +/- 0.6 pmol/L; P < .05). Exercise or no exercise did not alter the insulin response to the OGTT for either group. Despite a higher glucose response to the glucose load in T2D, an acute exercise bout (single or multiple bouts) did not appear to alter glucose control the following day in either the individuals with type 2 diabetes or the control group.

Replication defective viral vectors provide a potentially useful means of gene transfer to oligodendrocytes and thus for studying the pathogenesis of white matter disease. In this study we have examined the expression pattern of E1/E3 deleted adenoviral vectors expressing the reporter gene LacZ (AdlacZ) as a means of establishing the value of these vectors for gene delivery to oligodendrocytes in adult rat white matter. Our results indicate that although such an approach can be used to induce transgene expression in oligodendrocytes, it is complicated by both immunogenic and cytopathic effects. Thus, in normal animals, injection of DeltaE1/E3 adenoviral vectors was associated with a robust immune response that led to a lack of expression by 40 days after injection. In order to overcome this complication, virus was injected into the white matter of immuno-deficient athymic rats. These experiments indi- cated that even in the absence of a T cell response high viral titres of DeltaE1/E3 adenoviral vectors had a profound cytopathic effect leading to death of oligodendrocytes and hence demyelination. A similar cytopathic effect was demonstrated using an adenoviral vector expressing the neurocytokine ciliary neurotrophic factor (AdCNTF). As the titre of injected virus was decreased there was a significant decrease in the number of transgene expressing cells. These experiments therefore indicated that in immunodeficient recipients there is a narrow window of virus titre that results in a high rate of infectivity and expression without significant cytopathic consequences. At higher viral titres the cytopathic effects include oligodendrocyte death and demyelination, while at lower titres there is a significant decrease in the efficiency of the number of cells expressing the transgene.

PfPK4, a protein kinase gene from the human malarial parasite Plasmodium falciparum, has been cloned utilizing oligonucleotide probing. The gene encodes a protein of a predicted length of 1123 amino acids, and within this amino acid sequence all the conserved regions characteristic of protein kinases can be identified. The catalytic kinase domain possesses highest identities (34-37%) with eukaryotic initiation factor-2alpha (eIF-2alpha) kinases, especially haem-regulated inhibitory (HRI) protein kinases. There are two kinase inserts in PfPK4, located at positions common to eIF-2alpha kinases. The first insert separates kinase subdomains IV and VI by 559 amino acids, and the second subdomains VII and VIII by 41 amino acids. Both inserts are larger than their homologues in eIF-2alpha kinases. The sequence of PfPK4 has one putative haemin-binding site. The recombinant protein, expressed in Escherichia coli, phosphorylates a synthetic peptide representing a substrate of eIF-2alpha kinases. Autophosphorylation and substrate phosphorylation are inhibited by haemin. Thus PfPK4 appears to be the first protozoan protein kinase related to eIF-2alpha kinases and might be the first non-mammalian HRI kinase. Western blots indicated that the protein is expressed as major forms of 80 and 90 kDa. Whereas the 80 kDa form is present throughout the intraerythrocytic development and in merozoites, the two 90 kDa forms are only found in mature parasites. One of the latter is also present in the membrane fraction of erythrocytes harbouring segmenters. Confocal microscopy detected the protein distributed throughout the trophozoite, whereas it was found in discrete foci (punctate distribution) in segmenters. PfPK4 co-localizes with P. falciparum 83 kDa antigen/apical membrane antigen-1 at the apical complex in segmenters and merozoites, but does not co-localize with rhoptry-associated protein-1.

A mitogen-activated protein (MAP) kinase gene, PfMAP, from Plasmodium falciparum was recently identified. We expressed this gene in Escherichia coli to test whether it encodes a functional MAP kinase. Recombinant PfMAP kinase autophosphorylates on both the tyrosine and threonine residues within the TXY motif, and readily phosphorylates myelin basic protein as exogenous substrate. This identifies the PfMAP gene product as a true member of the growing family of MAP kinases. Wild-type PfMAP kinase expressed in COS-7 (SV40 transformed African green monkey kidney) cells seemed to induce apoptosis in these cells. Western blots and immunoprecipitations indicated that the kinase is expressed during the growth of the parasite in the red blood cell as three major forms: truncated forms with apparent molecular masses of 40 kDa and 80 kDa, and as a protein of approximately 150 kDa. The 40 kDa form is present throughout the intraerythrocytic development, whereas the two larger forms are only detected in mature parasites. The 40 kDa and 80 kDa forms are tyrosine phosphorylated, indicating that they represent the active forms of the PfMAP kinase. The total PfMAP kinase activity constantly increases with the maturation of the parasite.

In the course of our studies on cell cycle regulation mechanisms of Plasmodium falciparum, we investigated expression pattern, kinase activity, and localization of PfPK5, a putative malarial member of the family of cyclin-dependent protein kinase (cdks). The kinase was immunoprecipitated from parasites of selected stages and from parasites blocked with the cell-cycle inhibitor aphidicolin. An elevated kinase activity of PfPK5 from aphidicolin-blocked cells suggested that the enzyme might be implicated in the regulation of the parasite's S-phase. To further investigate this hypothetical function, parasite cultures were treated with the specific cdk inhibitors flavopiridol and olomoucine, which act on PfPK5 in vitro at similar concentrations as on other cdks. When applied during the nuclear division cycles of the parasite, both drugs markedly inhibited the DNA synthesis, as predicted from our proposition that PfPK5 is necessary to activate or maintain the parasite S-phase. Immunolocalization studies provide further evidence for this potential role of PfPK5.

Recently, glutathione reductase (GR) has emerged as a promising target for antiparasitic drugs. The central role of GR in cellular antioxidant defence, the particular susceptibility of intracellular parasites like Plasmodium falciparum to oxidative stress, and successful inhibitor studies substantiate this approach. However, more information is required on the structural and functional characteristics of GR from malarial parasites and differences from the enzyme of host erythrocytes. We have identified a putative P. falciparum GR gene coding for a polypeptide (PfGR2) of 500 amino acids that exhibits 40-45% sequence identity with GR enzymes from other species. 18 out of 19 residues contributing to glutathione binding are identical in the putative PfGR2 and human GR. According to Southern blot analysis, the PfGR2 gene is present as a single-copy gene. It is expressed during the intraerythrocytic life cycle. Stage-specific Northern blot analysis demonstrates that the PfGR2 gene is only weakly transcribed in ring, early trophozoite, and segmenter stages; major transcription occurs in the late trophozoite/early schizont stage. This is consistent with the high glutathione reductase activity found in early schizonts. Other data also suggest that PfGR2 corresponds to the enzyme isolated from parasitized erythrocytes. These criteria include the subunit molecular mass (56.2 kDa), the N-terminal sequence (VYDLIVIGGGSGGMA), the presence of specific sequence motifs at ligand-binding sites, and, as demonstrated by Western blotting, the occurrence of a unique chain segment in the core of the central domain. In view of these data, the function(s) of PfGR2 as well as PfGR1, the product of another GR-like gene of P. falciparum (Müller et al., 1995) should be carefully assessed.

The immunosuppressive drug cyclosporin A (CsA) inhibits the growth of malaria parasites in vitro and in vivo. Cyclosporin A exerts its immunosuppressive effect in T lymphocytes by binding to cyclophilin (CyP), a peptidylprolyl cis-trans isomerase (PPIase). It is believed that the cyclosporin/cyclophilin complex inhibits a Ca(2+)-activated protein phosphatase, calcineurin, involved in T-cell activation. A cDNA encoding a cyclophilin of the human malaria parasite Plasmodium falciparum has been isolated as a step in the elucidation of the mechanism of antimalarial action of CsA. This cDNA, termed PfCyP, encodes a protein of 195 amino acids which has highest similarity with the Candida albicans (73.1%) and the Drosophila melanogaster (73.1%) cytoplasmic cyclophilins. A Northern blot reveals an approximately 900-bp nucleotide transcript that is consistent with the predicted size of the encoded polypeptide. The predicted PfCyP protein has a putative endoplasmic-reticulum-directed signal sequence at its N-terminus and two potential N-linked glycosylation sites. Expression of PfCyP RNA in an in vitro translation/translocation system reveals that the PfCyP protein is translocated across microsomes, that the signal peptide is cleaved and that the PfCyP protein is glycosylated at two sites. The PfCyP cDNA open reading frame coding for the predicted mature protein has been expressed in Escherichia coli. The purified recombinant protein is an active PPIase (kcat/Km = 2.3 x 10(6) s-1 M-1); this enzymic activity is inhibited by CsA (IC50 = 10 nM). The PfCyP protein has thus the same sensitivity to CsA as the PPIase activity associated with P. falciparum extracts [Bell, A. et al. (1994) Biochem. Pharmacol. 48, 495-503] suggesting that PfCyP may be responsible for the PPIase activity in those extracts. If different cyclophilins exist in P. falciparum, we conclude that either the PfCyP protein is the major cyclophilin detected in the parasite or that there are other cyclophilins with similar susceptibilities to CsA.

A protein kinase gene (PfPK1) has been isolated from the human parasite Plasmodium falciparum by using a mixed oligonucleotide pool which corresponds to a highly conserved region of serine/threonine protein kinases. The gene, which contains one intron, encodes a protein with a predicted length of 909 amino acids. The predicted protein contains all the conserved sequences characteristic of a protein kinase catalytic domain. These sequences are discontinuous, however, since they are separated by two large kinase inserts with 178 and 330 amino acids in size. Specific antisera were raised against recombinant fragments of the protein and a PfPK1-specific peptide. Using one of these antibodies, a functional protein kinase was precipitated from malarial lysates and this kinase recognized casein as an exogenous substrate. PfPK1 was expressed in a stage-specific fashion and also had a stage-specific cellular localization. During the intraerythrocytic life cycle, PfPK1 shifts from the parasite cytosol to the parasite membrane fraction. An unusual feature of PfPK1 is its electrophoretic mobility on SDS-PAGE. Whereas the predicted protein size is about 100 kDa, the apparent size is about 70 kDa. There are no indications for RNA processing and we could exclude proteolytic processing as an explanation.

Microtubule inhibitors are active against the human malarial parasite Plasmodium falciparum, but whether these drugs actually interact with parasite tubulins is not known. It has not previously been possible to produce mg quantities of isolated, soluble tubulin subunits for drug-binding experiments. A cDNA encoding P falciparum beta-tubulin was expressed and the protein secreted in Bacillus brevis. With the addition of EGTA to the culture medium, which increases shedding of proteins from the cell surface, up to 2 mg/l recombinant beta-tubulin was obtained in supernatants. It is not clear why B brevis is able to secrete this normally cytoplasmic protein, but the secretion levels of recombinant proteins may be related to the net charge of the first few residues of the mature polypeptide.

We have cloned a Plasmodium falciparum gene that belongs to the nuclear Tat-binding protein (TBP) gene family. This gene, PfTBP, is (A + T)-rich and encodes a 49.5-kDa protein. The predicted protein encoded by this gene has highest similarity to the slime mold protein DdTBP10 (86%) and to the yeast protein SUG1 (81.8%), both of which belong to the Tat-binding protein family. In agreement with the characteristics of this family, PfTBP contains a highly conserved domain of approximately 200 amino acids, in which are found the motifs A and B of ATPases, and amino acid sequences characteristic of a large family of RNA or DNA helicases, suggesting a role in RNA or DNA unwinding. Like DdTBP10, the PfTBP protein has a heptad repeat of four leucine residues, reminiscent of a leucine zipper motif known to mediate dimerization. We have further characterized PfTBP gene expression by Northern-blot analysis. This gene is expressed in a stage-specific manner, with higher expression in the late trophozoite stage. The recombinant PfTBP gene has been expressed in Escherichia coli and a polyclonal antiserum has been raised in rabbits against the recombinant protein. This antibody has been used to study the protein in the parasite. The gene product is expressed in a stage-specific manner with higher expression in the late trophozoite and schizont stages, and is localized in the nucleus of the erythrocytic stage parasite. Thus the protein might have a function in DNA synthesis and/or in transcription, as is the case for other Tat-binding proteins.

The immunosuppressive peptide cyclosporin A inhibits the growth of malaria parasites in vitro and in vivo, but little is known about its mechanism of antimalarial action. The immunosuppressive action of cyclosporin A is believed to result from binding of the drug to cyclophilins (intracellular peptidyl-prolyl cis-trans isomerases), and inhibition of the protein phosphatase calcineurin by the cyclosporin A-cyclophilin complex. Two immunosuppressive macrolides, FK506 and rapamycin, bind to a distinct isomerase, FKBP12, and the FK506-FKBP complex also inhibits calcineurin. Calcineurin itself is apparently involved in signal transduction between the T-cell membrane and nucleus, and its inhibition blocks T-cell activation. Rapamycin inhibits a later step in T-cell proliferation. Peptidyl-propyl cis-trans isomerase activity was detected in extracts of Plasmodium falciparum. It was completely inhibited by concentrations of cyclosporin A above 0.1 microM, but not by FK506 or rapamycin, and probably represented one or more cyclophilins. Comparison of the antimalarial and anti-isomerase activities of a series of cyclosporin analogues failed to reveal a correlation between the two properties. Cyclosporin A and its more active 8'-oxymethyl-dihydro-derivative, in combination with the cyclophilin-containing P. falciparum extract, inhibited the protein phosphatase activity of bovine calcineurin. Therefore inhibition of a putative P. falciparum calcineurin by a complex of CsA and cyclophilin might be responsible for the antimalarial action of the drug. The most active cyclosporin, however, was a 3'-keto-derivative of cyclosporin D (SDZ PSC-833) which inhibited P. falciparum growth with a 50% inhibitory concentration (IC50) of 0.032 microM (compared with 0.30 microM for cyclosporin A), but was a poor inhibitor of the parasite isomerase. 3'-Keto-cyclosporin D has negligible immunosuppressive activity, but it strongly inhibits the P-glycoprotein of multi-drug resistant mammalian tumour cells. FK506 and rapamycin were also active antimalarials (IC50 of 1.9 and 2.6 microM, respectively) but in the absence of detectable FKBP in P. falciparum extracts, their mechanisms of antimalarial action remain unclear.

The unusual Ca(2+)-dependent protein kinase from Plasmodium falciparum (PfCPK) [1], whose gene structure and expression in bacteria have been reported [1], was purified to homogeneity. The purified recombinant kinase has a native molecular mass of 62,000, is activated by Ca2+ (K0.5 = 15 microM) in the presence of Mg2+ or Mn2+, and can associate with 45Ca2+. The activation by Ca2+ could be partially replaced by Mn2+, but not by Zn2+ or Mg2+. PfCPK preferentially phosphorylated casein and histone H1. The Km and Vmax for Mg2+ ATP were 26 microM and 70 nmol min-1 mg-1, respectively, with casein as substrate; and 34 microM and 143 nmol min-1 mg-1, respectively, with histone H1 as substrate. The kinase undergoes autophosphorylation on both serine and threonine residues. Calmodulin antagonists (calmidazolium, trifluoperazine, N-[6-aminohexyl]-5-chloro-1-napthalene-sulfonamide, and ophiobolin A) could inhibit the kinase activation, but much higher concentrations of the antagonists are needed than was required to inhibit calmodulin-mediated effects. PfCPK preferentially phosphorylates proteins of the host erythrocytic membrane in vitro but phosphorylates parasitic proteins only to a minor extent. The selectivity of the phosphorylation may be partially controlled by phosphatidylserine which is bound to some of the erythrocytic membrane proteins. Using a rabbit polyclonal antiserum against the recombinant protein, the kinase was found to be mainly expressed in the ring and schizont stages, and mainly localized in the parasitic membrane-organelle fraction and partially localized on the erythrocytic membrane.

Calcium-dependent protein kinase from Plasmodium falciparum (PfCPK) is a multidomain protein composed of an N-terminal kinase domain connected via a linker region to a C-terminal CaM-like calcium-binding domain. The kinase can be activated by Ca2+ alone and associates with 45Ca2+. Here we describe the calcium-binding properties of the kinase and the significance of the individual calcium-binding sites with respect to enzymatic activation, as well as the Ca(2+)-induced conformational change as detected by circular dichroism. As predicted from the cDNA sequence, the kinase has four EF-hand calcium-binding sites in the C-terminal domain. To understand the roles of the individual calcium-binding sites, two series of mutations were generated at the individual EF-hand motifs. The highly conserved glutamic acid residue at position 12 in each calcium-binding loop was mutated to either lysine or glutamine, and therefore a total of eight mutants were generated. Either of these mutations (to lysine or glutamine) is sufficient to eliminate calcium binding at the mutated site. Sites I and II appear to be crucial for both Ca(2+)-induced conformational change and enzymatic activation. Whereas mutations at site II almost completely abolish kinase activity, mutations at site I are also deleterious and dramatically reduce the sensitivity of the Ca(2+)-induced conformational change and the Ca(2+)-dependent activation. Mutations at sites III and IV have minor effects.

Two major phosphoproteins of Plasmodium falciparum could be identified by partial amino acid sequencing as the plasmodial members of the hsp 70 heat shock protein family, Pfhsp and Pfgrp. According to phosphoamino acid analyses of Pfhsp and Pfgrp isolated from [32P]orthophosphate-labeled malarial cultures, both proteins were phosphorylated in Ser and Thr. While Pfhsp contains higher amounts of labeled phosphoserine, Pfgrp contains higher amounts of phosphothreonine. Phosphorylation of both proteins increased throughout the entire erythrocytic growth cycle. At the trophozoite and schizont stages Pfhsp and Pfgrp are the most prominent phosphoproteins of Plasmodium falciparum. Using multiply redundant oligonucleotides directed against the N-terminus of Pfgrp we cloned and sequenced the entire Pfgrp gene. The gene encodes a product with a predicted length of 652 amino acids. The deduced amino acid sequence has identities of 65.5% and 65.0% to the human and rat grp78 proteins, respectively. Pfgrp possesses a classical N-terminal leader sequence. The published grp78 related gene sequences of Plasmodium falciparum are all fragments of the same plasmodial gene.

Complications may arise from the failure to remove a dislocated intraocular lens, even when it appears to rest in a benign position and pose no problems. We examined a patient with a second intraocular lens left in the inferior vitreous cavity. Shifting the loose lens resulted in eventual complications and visual disability; removing the dislocated lens and replacing the functional intraocular lens yielded good results.

Purpose: To determine whether lacrimal gland acinar cells are involved in modulating local immune response; to isolate and characterize a lacrimal gland factor that exerts biological activities on T lymphocytes., Methods: A protein factor has been purified from lacrimal gland extracts by a combination of ion exchange and gel-filtration chromatography. This factor has the capacity to enhance proliferation of T lymphocytes upon stimulation with a mitogen or an antigen. We have, therefore, called this substance lacrimal gland-derived lymphocyte proliferation potentiating factor (LG-F)., Results: Lacrimal gland-derived lymphocyte proliferation potentiating factor has a molecular weight of approximately 65,000 daltons as determined by gel-filtration and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. T cells demonstrate greater proliferation when cultured with high concentrations of concanavalin A (Con A) in the presence of LG-F, as compared with culture without addition of LG-F. This enhancing effect of LG-F may be mediated by IL-2, because the final cell count correlates with the levels of IL-2 secreted by LG-F-activated cells. Lacrimal gland-derived lymphocyte proliferation potentiating factor is nonmitogenic for T cells, but its potentiating effect is antigen-dependent. Dual stimulation of OA-primed T cells with both OA and LG-F results in greater proliferative activity, in contrast to culture with OA alone., Conclusions: The results suggest that lacrimal gland cells may interact with the immune system by elaborating nonspecific factors that modulate lymphocyte proliferation and augment lymphokine production. The presence of such a factor in the lacrimal gland may prove to be of importance in the generation of local immune responses.

An unusual protein kinase gene, termed PfCPK, was isolated from Plasmodium falciparum. The gene, which contains five exons and four introns, encodes a product with a predicted length of 524 amino acids. The amino-terminal segment of the predicted protein contains all of the conserved sequences characteristic of a protein kinase catalytic domain and has a high homology to several protein serine-threonine kinase subfamilies (30-41% amino acid identities). These subfamilies include calcium/calmodulin-dependent protein kinases, calcium-dependent protein kinase, ribosomal S6 protein kinase, cyclic nucleotide-dependent protein kinases, protein kinase C, and the yeast SNF1 subfamily. All of these protein kinases are relatively close in the phylogeny tree and within the kinase catalytic domains have about 35% amino acid identities to each other, suggesting that PfCPK is also in this region of the phylogeny tree. An unusual feature of PfCPK is that its carboxyl-terminal segment displays homology to the EF hand calcium-binding proteins, for example 34% amino acid identity to chicken fast skeletal muscle troponin C and 35% amino acid identity to human calmodulin. Like troponin Cs and calmodulins, PfCPK also contains four EF hand calcium-binding motifs. Furthermore, the four introns in the PfCPK gene are all located in the carboxyl-terminal putative EF hand calcium-binding region (EF hand calcium-binding proteins from higher eukaryotes generally contain multiple introns). This combination of a protein kinase and an EF hand calcium-binding protein in a single polypeptide implies that PfCPK may be directly activated by calcium. Constructs containing the full-length PfCPK cDNA have been expressed in Escherichia coli at a high level to generate a 60-kDa recombinant protein. Compared with similar fractions from control cells, the fraction containing PfCPK recombinant protein exhibited an elevated protein kinase activity which was Ca(2+)-dependent.

At low concentrations, both isomers of tubulozole (C, T) inhibit Plasmodium falciparum but only tubulozole C inhibits mammalian cells. Since tubulozole C prevents polymerization of mammalian tubulin whereas tubulozole T does not, the antimalarial action of tubulozoles may not involve microtubules. The present study concerns the inhibition of parasite protein synthesis by the tubulozoles. While tubulozoles took 3-4 h to kill parasites in erythrocytic culture, they inhibited protein synthesis within 10 min. The concentrations of the drug required were, however, too high for this to account for their antimalarial action. The microtubule inhibitor colcemid inhibited protein synthesis rapidly and at relevant concentrations, but vinblastine did not inhibit protein synthesis. Tubulozole T and colcemid inhibited protein synthesis posttranscriptionally since they had little effect on RNA synthesis. Analysis of labelled parasite proteins by two-dimensional gel electrophoresis showed that while it inhibited synthesis of most proteins to the same degree, tubulozole T super-inhibited the synthesis of certain proteins. This may cause its antimalarial effect at low concentrations.

Three tryptic-peptide sequences of an 88-kDa pair of phosphoproteins of the malaria parasite Plasmodium falciparum were determined. They exhibit a striking similarity to corresponding sequences of the 89-kDa domain of human erythrocyte ankyrin. [35S]Methionine labeling of the two proteins demonstrated their parasitic origin. Using an appropriate oligonucleotide probe, Southern-blot analysis of genomic malaria DNA and Northern-blot analysis of malaria RNA suggest the existence of ankyrin-related sequences in the parasite genome and the presence of an ankyrin-related transcript of about 3.2 kb. Our studies provide further evidence of malaria-specific analogues of host-cell proteins, implying an unusual kind of parasite/host interaction.

A putative protein kinase gene (PfPK2) has been isolated from the human parasite Plasmodium falciparum by using a mixed oligonucleotide pool which corresponds to a highly conserved region of serine/threonine protein kinases. The complete nucleotide sequence of 5 kb suggests the existence of a second transcriptional unit besides that of the PfPK2 gene, separated by a highly (A+T)-rich region and transcribed in a different orientation. No intron sequence exists in PfPK2. The predicted amino acid sequence of PfPK2 contains features characteristic of eukaryotic serine/threonine protein kinases. Within its putative catalytic domain it shares 33%, 30%, and 28% amino acid identities with rat calcium-calmodulin-dependent protein kinase, human protein kinase C, and bovine cAMP-dependent protein kinase, respectively. Outside the catalytic domain, however, PfPK2 has no homology with regulatory domains of other protein kinases, indicating PfPK2 might be modulated by signals different from those of higher eukaryotes or might be associated with other regulatory subunits. Using a specific antiserum raised in rabbits against a recombinant fragment of the protein expressed in Escherichia coli, PfPK2 was found to be expressed in a stage-specific fashion and mainly localized in the parasitic membrane.

Fifty-nine Plasmodium falciparum specific phosphoproteins with molecular weights between 15,000 and 192,000 were analyzed by SDS-PAGE and two-dimensional gel electrophoresis. 40 phosphoproteins were identified by [gamma-32P]ATP labeling of cell lysates, 19 by [32P]orthophosphate labeling of parasitic cultures in vivo. Changes in the phosphorylation pattern during the infectious erythrocytic cycle were determined for all proteins. In parallel, cell fractionation studies were done to follow up possible changes in the cellular distribution of these proteins. Several phosphoproteins are associated with the membrane fraction of infected erythrocytes. One pair of proteins of approx. 88 kDa and a pI of about 5.0 was further characterized. Both proteins are located in the parasitic fractions as well as in the membrane of infected erythrocytes during the entire cycle. Phosphorylation of these proteins, however, is restricted to the trophozoite and schizont stages. Peptide mapping studies demonstrated that both proteins are identical with the exception of minor modifications which are probably not the result of differences in phosphorylation.

In order to inhibit the in vitro translation of Plasmodium falciparum mRNA coding for the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS), oligodeoxynucleotides (ODNs) were directed against the translation initiation site or a site in the TS-coding region. In both cases considerable hybridization arrest, i.e. greater than 50% inhibition, was only achieved if the lengths of the ODNs to the two regions were 30 and 39 nucleotides, respectively, or longer. The ODN with the highest efficiency was a 49-mer directed against the TS-coding region (OTS49); 45 microM was sufficient to inhibit the expression of DHFR-TS by almost 90%. In this case the synthesis of DHFR-TS was interrupted at the binding site of OTS49 by a RNase H-independent mechanism. The resulting polypeptide was smaller (55 kDa) than one subunit of the native protein (71 kDa) and lacked TS activity.

Although several approaches to the control of human parasites are possible, the prevention and therapy of the corresponding diseases still remain a difficult task. The development of vaccines has been hampered by the poor immunological response to or the high variability of parasitic antigens. Problems also arise for chemotherapy where differences in the biochemistry of host and parasite must be exploited. The increasingly difficult search for new drugs is always challenged by the appearance of resistance.

The mode of action of the tubulozole isomers, recently recognized as a new class of potential antimalarial agents, was investigated. Whereas neither glycolysis, protease activity, or nucleic acid biosynthesis was primarily affected, protein biosynthesis decreased soon after addition of the drug. Inhibitors of protein biosynthesis, however, did not show synergistic activity with tubulozole. Colcemid, on the other hand, had an effect on protein synthesis similar to that seen with the tubulozoles. Furthermore, combinations of the tubulozole isomers with compounds known to interact with tubulin inhibited malaria in a synergistic or antagonistic fashion. Therefore, the inhibition might be elicited by interaction with tubulin or some other component of the microtubules. This is remarkable insofar as only one of the tubulozole isomers affects mammalian cells by binding to tubulin.

The majority of immunoglobulin in tears in various species is of the IgA isotype and is produced mainly by plasma cells of the lacrimal gland. The mechanism responsible for lodging of IgA committed cells in lacrimal gland is unknown, but could occur as a result of retained T helper cells in the lacrimal gland. The present experiments use an in-vitro adherence assay to examine the interaction of various murine lymphocyte populations with frozen sections of mouse lacrimal gland. Splenic and Peyer's Patch lymphocytes, as well as T-cell enriched and T-cell depleted lymphocyte populations were incubated with thin sections of lacrimal gland. Adherent lymphocytes were counted in randomly selected fields for each experiment. Each lymphocyte population was tested at least three times. It was found that more Peyer's Patch cells adhered to lacrimal gland than did spleen cells, and this increase was due to T-cells. The T-cell population responsible for adherence was localized to the helper T-cell population which appeared greater with Peyer's Patch T helper cells than splenic T helper cells. The data suggest that a lacrimal gland-T-cell interaction could retard the migration of a subpopulation of T helper cells as they migrate through the lacrimal gland. It is speculated that such T helper cells could be responsible for the terminal differentiation of IgA B cells to plasma cells, thus explaining the predominance of IgA plasma cells in the lacrimal gland.

Tumour promoter phorbol myristate acetate (PMA) stimulates and causes proliferation of cells from the spleen, thymus and peripheral blood of Xenopus laevis, the clawed toad. Phagocytic cells become activated and from foci of clustered cells. B cells blast and change membrane immunoglobulin (Ig) expression; a small portion go on to secrete Ig. The surface Ig-negative splenocyte population, which contains T cells, responds to PMA by active proliferation. Splenocytes, once stimulated by PMA, become refractory to further addition of PMA but respond instead to ConA. These results are discussed in the general context of the effect of PMA lymphocytes.

A 31-year-old man with myopia underwent bilateral radial keratotomy. Except for a microperforation of the cornea of the left eye, the operative procedures were uneventful. The postoperative uncorrected visual acuity was excellent in both eyes until a cataract suddenly developed in the left eye 3 1/2 months after the procedure. No preexisting ocular or systemic conditions existed to explain the sudden development of the cataract, and no evidence of iris or anterior capsular damage could be seen on examination. The sudden development of this cataract after radial keratotomy remains unexplained.

A subsample was drawn from an earlier collection of data in order to answer a number of questions related to "acid" (LSD, STP) flashbacks. Acid users who reported flashbacks also reported significantly more marijuana use than those who did not; the two groups did not differ on use of other drugs, including acid. Simple correlations and multiple regression analyses both showed extent of marijuana use to be the only drug variable significantly related to acid flashbacks. No optimal combination of marijuana and acid improved flashback prediction. Among acid users, correlations between amounts of use for various drugs were high and significant, excepting only the marijuana-acid correlation.

Polyclonal activation of T-cells with concanavalin A has been used as an in vitro test system to study immunosuppression induced by the avian retrovirus MAV-2-O. The mitogenic responsiveness of peripheral blood lymphocytes from infected chickens was only weakly suppressed, that of spleen lymphocytes was highly suppressed. Addition of conditioned media rich in T-cell growth factor (TCGF) activity to cultures of infected birds resulted in a reconstitution of the suppressed mitogenic responsiveness up to the level found in lymphocyte cultures from normal chickens. This indicates that the defect of the observed immunopathology is not at the level of responding T-cells. Measurements of TCGF production always showed reduced TCGF levels in the suppressed cultures suggesting that there is a defect at the TCGF production level. This is further supported by the failure to reconstitute the suppressed mitogenic response with normal macrophages, which are involved in activation of TCGF producer T-cells.

Avian myeloblastosis-associated virus-induced nephroblastomas are tumors consisting mainly of mesenchymal and epithelial renal elements with variable degrees of differentiation. The spatial distribution of developmental stages reflects a gradient of differentiation from less differential structures in the periphery towards more differentiated structures in the center of the lobules formed in the nephroblastomas. These heterogenic tumors contain discrete virus-cell DNA junction fragments and are therefore clonal outgrowths of a single transformed cell. These findings support the hypothesis that a mesenchymal, nephrogenic cell residual in the postembryonic kidney is the origin of the tumor, which grows by proliferation and differentiation of this target cell. All the tumors expressed higher levels of viral genomic and env messages than nontransformed tissue from the same kidney. A screening of oncogene expression with 13 different oncogenes revealed enhanced myc levels. There was, however, no rearrangement of c-myc or of the other oncogenes detected with EcoRI-digested tumor DNAs. This suggests that there is no insertion of viral elements adjacent to a c-myc. The levels of myc expression in embryonic kidneys were as high as in the tumors. Therefore, the enhanced myc expression in nephroblastomas is a reflection of the embryonic status of the tumor rather than a newly acquired function. This finding, plus the similarity of development and morphology of nephroblastomas and embryonic kidneys, suggests that the tumors arise as a result of a deficiency in a function which turns the embryonic status off.

In order to determine the proteins of major allergenic importance in honeybee venom (Apis mellifera) it was chromatographed on G-50 Sephadex. The four major protein peaks eluted were identified as hyaluronidase, phospholipase, melittin, and apamin. Testing these preparations on the leukocytes of 6 honeybee-sensitive patients, with the in vitro method of histamine release, revealed that all individuals were most sensitive to phospholipase A. IgE antibodies against phospholipase A (RAST) were found in the sera of honeybee-sensitive patients and IgG antibodies to this venom component were found in the sera from beekeepers and venom-treated patients. Melittin appeared to be allergenic in several patients, but the results were variable and were possibly due to contamination with phospholipase. All patients were insensitive to the hyaluronidase and apamin preparations. We conclude that phospholipase A is the major allergen of honeybee venom and, since this protein is readily available, it should be useful for diagnostic and therapeutic studies as well as for the standardization of materials used in the management of honeybee-sensitive patients.

The lipid-containing bacteriophage PM2 was dissociated stepwise in 1 M NcCl (pH 7.2)with increasing urea concentrations. In 2 M urea the following substructures could be identified: (a) a nucleocapsid containing all of the viral lipid, the DNA, proteins III and IV, plus a fraction of protein II, and (b) a second substructure respresenting particles which contained all viral elements except protein I, the spike protein. In 4 M urea the viral nucleocapsid containing all of proteins III and IV, the DNA, plus a fraction of protein II, was isolated. Upon increasing the urea concentration further, this nucleocapsid is stable up to 8.5 M urea; in 9 M urea protein III was partly dissociated from the nucleocapsid. The nucleocapsid in 4--8.5 M urea is stabilized by the addition of 0.1--3 M NaCl but dissociates if the NaCl concentration is less than 0.1 M. The nucleocapsid was also dissociated in 4--8.5 M urea at pH 4.5. The nucleocapsid structures and some intermediate morphological subunits have been analysed by physical methods, enabling us to draw some conclusions about the structure and hydration of the virus.

Plasma cells containing intracellular inclusions of immunoglobulin (Russell bodies) are known as Mott cells, and are found in large numbers in lymphoid organs in autoimmune mice. Hybridoma technique was used to produce cell lines of this phenotype by fusing spleen cells from a NZB mouse with a nonproducing hybridoma cell line (Sp2/0-Ag14), allowing us to carry out studies of this cell type at the biochemical level. Ultrastructurally the inclusions were distended cisternae of rough endoplasmic reticulum, suggesting a block in the secretory pathway of the cells. Biosynthetic labeling studies confirmed that these cell lines have either a complete or partial block of secretion of immunoglobulin, possibly due to an abnormal light chain.

A specialized lymphoid tissue in rabbit conjunctiva was studied by various histologic and immunologic techniques and compared with similar structures along other mucosal surfaces. The flattened conjunctival lymphoepithelium overlying the lymphoid follicles was devoid of goblet cells. This lack of goblet cells is characteristic of epithelium overlying similar lymphoid collections in gut and bronchus. The lymphoid follicles demonstrated neither intra- nor extracellular immunoglobulin, and the lymphocytes in these follicles were composed of B-cells and T-cells, when studied by various immunologic techniques. A high proportion of these lymphocytes showed surface immunoglobulin A (IgA), and a high proportion of IgA precursors were determined by pokeweed mitogen (PWM) stimulation in 4-day cultures. The morphologic and immunologic results are similar to those obtained from gut and bronchus, tissues known to disseminate lymphoid cells to other mucosal sites already committed to antigen and IgA isotype. It is speculated that conjunctival associated lymphoid tissue of rabbit is part of a generalized system of secretory immunity capable of sampling conjunctival applied antigen, and then disseminating cells committed to IgA antibody production to other mucosal sites.

The popular azure-eosin stain of Nocht has been reformulated to provide consistent, intense staining of semithin sections of water-miscible methacrylate embedded tissues.

The induction of caprine keratoconjunctivitis by the subconjunctival inoculation of a cloned culture of Mycoplasma conjunctivae is described. The clinical course of the experimental disease was similar to that noted in naturally occurring outbreaks of "pink-eye" among goats, and biopsies of inflamed conjunctivae showed similar histological response. M. conjunctivae was consistently recovered from the inflamed conjunctival tissues of inoculated animals that developed ocular disease, thus fulfilling Koch's postulates and establishing this organism as an etiological agent of caprine keratoconjunctivitis. Immunological studies suggested that cellular immune mechanisms may play a role in protecting animals from disease produced by this mycoplasma.