Molecular basis for immunoglobulin M specificity to epitopes in Cryptococcus neoformans polysaccharide that elicit protective and nonprotective antibodies.

The protective efficacy of antibodies (Abs) to Cryptococcus neoformans glucuronoxylomannan (GXM) is dependent on Ab fine specificity. Two clonally related immunoglobulin M monoclonal Abs (MAbs) (12A1 and 13F1) differ in fine specificity and protective efficacy, presumably due to variable (V)-region sequence differences resulting from somatic mutations. MAb 12A1 is protective and produces annular immunofluorescence (IF) on serotype D C. neoformans, while MAb 13F1 is not protective and produces punctate IF. To determine the Ab molecular determinants responsible for the IF pattern, site-directed mutagenesis of the MAb 12A1 heavy-chain V region (V(H)) was followed by serological and functional studies of the various mutants. Changing two selected amino acids in the 12A1 V(H) binding cavity to the corresponding residues in the 13F1 V(H) altered the IF pattern from annular to punctate, reduced opsonic efficacy, and abolished recognition by an anti-idiotypic Ab. Analysis of the binding of the various mutants to peptide mimetics revealed that different amino acids were responsible for GXM binding and peptide specificity. The results suggest that V-region motifs associated with annular binding and opsonic activity may be predictive of Ab efficacy against C. neoformans. This has important implications for immunotherapy and vaccine design that are reinforced by the finding that GXM and peptide reactivities are determined by different amino acid residues.

Human and murine immunoglobulin expression vector cassettes.

We describe the construction of new immunoglobulin (Ig) expression vectors and their use in the production of recombinant chimeric Ig molecules in transfected mammalian cells. The vectors contain the cDNA encoding the constant regions of human (mu, alpha1, gammal, gamma2, gamma3, gamma4, kappa) and murine (mu, gamma2a, kappa) Ig heavy and light chains. Unique restriction sites flanking the Ig variable region allow for replacement of variable regions generated by PCR. The CMV promoter allows for the transfection and expression of Ig in non-lymphoid cells. Distinct drug selection markers for heavy chain and light chain expression vectors allows for sequential or co-transfection of the vectors. We show that secretion of recombinant Ig can reach 1.2 microg/ml per million cells per day for transfected B cells. Replacement of the variable region results in the production of functional Ig retaining antigen specificity.

Age-related difference in cardiac adaptation to chronic hypertension in rats, with and without nifedipine treatment.

UNLABELLED: Three myosin isozymes, V1 (alphaalpha MHC = Myosin Heavy Chain gene), V2 (alphabeta MHC) and V3 (betaalpha MHC) that are identified in the cardiac ventricles of most mammals have been shown to shift to a V3 predominance pattern during cardiac growth and in response to left ventricular pressure overload, and to V1 predominance following anti hypertensive treatment. This study examined whether long-term hypertension impairs the ability of the adult heart to restructure myosin isozyme proportions. Using pyrophosphate gel electrophoresis, we studied proportions of cardiac myosin isozymes (V1 and V3) in young (16 weeks) and adult (36 weeks) spontaneously hypertensive rats (SHR), and following 12 weeks of nifedipine (N) treatment in age-matched SHR rats (SHR-N). The values of V1 and V3 myosin isozymes were derived by adding half of the value of V2 to each isozyme proportion. The V3 proportion in the young SHR control (SHR-C) group (49%) was 34% higher (p < 0.05) than in the young Wistar Kyoto control (WKY-C) group (37%). However, the proportion was similarly high, though not statistically significant, in both the adult SHRC (73%) and WKY-C (71%) groups. The proportion in the young SHR-N group (29%) was 41% lower (p < 0.05) than in the young SHR-C group (49%), and the proportion in the adult SHR-N group (47%) was 34% lower (p < 0.05) than in the adult SHR-C group (73%). The ratio of left ventricular weight to body weight (LVW/BW), which determines left ventricular hypertrophy (LVH), was higher in both young and adult SHR-C (26%, p < 0.05, and 42%, p < 0.05, respectively) than in WKY-C groups. The mean LVW/BW was 27% (p < 0.05) greater in adult than in young SHR-C rats. The LVW/BW in both age groups of treated SHR-N was similar to that in age matched WKY-C rats. CONCLUSION: Our study showed that a rise in the V3 level occurs in young hypertensive rats, but no rise occurs in the V3 level in adult hypertensive rats. High blood pressure seems to contribute to the high V3 level in young hypertensive rats, but in adult hypertensive rats, high blood pressure does not accentuate the V3 rise already acquired due to the aging process. Nifedipine treatment in both young and adult hypertensive rats prevented the V3 rise due to hypertension and to the aging process. This effect of nifedipine seems to be through its antihypertensive action.

Experimental diabetes is associated with functional activation of protein kinase C epsilon and phosphorylation of troponin I in the heart, which are prevented by angiotensin II receptor blockade.

A cardiomyopathy that is characterized by an impairment in diastolic relaxation and a loss of calcium sensitivity of the isolated myofibril has been described in chronic diabetic animals and humans. To explore a possible role for protein kinase C (PKC)-mediated phosphorylation of myofibrillar proteins in this process, we characterized the subcellular distribution of the major PKC isoforms seen in the adult heart in cardiocytes isolated from diabetic rats and determined patterns of phosphorylation of the major regulatory proteins, including troponin I (TnI). Rats were made diabetic with a single injection of streptozotocin, and myocardiocytes were isolated and studied 3 to 4 weeks later. In nondiabetic animals, 76% of the PKC epsilon isoform was located in the cytosol and 24% was particulate, whereas in diabetic animals, 55% was cytosolic and 45% was particulate (P < .05). PKC delta, the other major PKC isoform seen in adult cardiocytes, did not show a change in subcellular localization. In parallel, TnI phosphorylation was increased 5-fold in cardiocytes isolated from the hearts of diabetic animals relative to control animals (P < .01). The change in PKC epsilon distribution and in TnI phosphorylation in diabetic animals was completely prevented by rendering the animals euglycemic with insulin or by concomitant treatment with a specific angiotensin II type-1 receptor (AT1) antagonist. Since PKC phosphorylation of TnI has been associated with a loss of calcium sensitivity of intact myofibrils, these data suggest that angiotensin II receptor-mediated activation of PKC may play a role in the contractile dysfunction seen in chronic diabetes.

Expression and regulation of mutant forms of cardiac TnI in a reconstituted actomyosin system: role of kinase dependent phosphorylation.

When phosphorylated, the inhibitory subunit of troponin (TnI) causes a loss in calcium sensitivity and a decrease in actomyosin ATPase. To examine this process, we bacterially expressed wild type TnI and TnI mutants in which serine 22 and 23, a putative protein kinase A (PKA) site, and threonine 143, a putative protein kinase C (PKC) site, were replaced by alanine S22A/23A and TI43A. PKA dependent phosphorylation was approximately 90% reduced in the S22A/23A mutant and unaffected in T143A. PKC dependent phosphorylation was markedly reduced in T 143A relative both to a wild type construct and to S22A/23A, although some residual phosphorylation (likely at sites other than T143) was seen. The calcium sensitivity (i.e. inhibition of actomyosin ATPase in the presence of EGTA) and regulation of the reconstituted actomyosin system was preserved in the absence of phosphorylation using wild type TnI or either mutant. Calcium sensitivity was decreased by both PKA and PKC with the wild type TnI but was unaffected by PKA when the S22A/23A mutant was employed and by PKC when the T143A mutant was reconstituted. The calcium dependency of the ATPase curve was substantially right shifted when PKC phosphorylated wild type TnI was employed for regulation, and this was markedly attenuated when T143 A was reassociated (although a slight rightward shift and a reduction in maximal ATPase activity was still seen). These data confirm that phosphorylation of TnI by regulatory kinases plays a major role in the regulation of myofibrillar ATPase. The N-terminal serines (22 and 23) appear to be uniquely important for the PKA response whereas threonine 143 is involved in the PKC response although other residues may also have functional significance.

Role of endogenous renin-angiotensin system in c-fos activation and PKC-epsilon translocation in adult rat hearts.

Myocardial stretch and the renin-angiotensin system have been implicated in the development of cardiac hypertrophy through the activation of specific target genes. However, the relative importance of these putative hypertrophic stimuli has not been established in vivo. We used an isolated isovolumic heart preparation in which coronary perfusion pressure (CPP), left ventricular end-diastolic pressure, and pharmacological therapy can be independently manipulated to study this relationship. High CPP (140 cmH2O), which increased coronary flow (8.99 vs. 17.6 ml/min) and left ventricular systolic pressure (50 vs. 91 mmHg), increased steady state c-fos mRNA expression 2.3-fold (all P < 0.01 vs. low CPP). In contrast, increased left ventricular end-diastolic pressure (25 mmHg) and/or infusion of angiotensin II in the absence of increased CPP was not associated with an increase in c-fos mRNA expression. The change in c-fos gene expression seen with increased CPP was largely reversed by treatment with an angiotensin type 1 (AT1) receptor blocker. Hearts perfused at high CPP demonstrated increased translocation/activation of protein kinase C-epsilon relative to controls. None of the hearts studied were ischemic during perfusion. Thus, in the perfused adult rat heart, dynamic, but not static, stretch activates the early response gene, c-fos, and may involve the endogenous reninangiotensin system and protein kinase C.

Troponin subunits contribute to altered myosin ATPase activity in diabetic cardiomyopathy.

Our group has documented that myocardial performance is impaired in the hearts of chronically diabetic rats and rabbits. Abnormalities in the contractile proteins and regulatory proteins may be responsible for the mechanical defects in the streptozotocin (STZ)-diabetic hearts. Previously, the major focus of our research on contractile proteins in abnormal states has concentrated on myosin ATPase and its isoenzymes. Our present study is based on the overall hypothesis that regulatory proteins, in addition to contractile protein, myosin contribute to altered cardiac contractile performance in the rat model of diabetic cardiomyopathy. The purpose of our research was to define the role of cardiac regulatory proteins (troponin-tropomyosin) in the regulation of actomyosin system in diabetic cardiomyopathy. For baseline data, myofibrillar ATPase studies were conducted in the myofibrils from control and diabetic rats. To focus on the regulatory proteins (troponin and tropomyosin), individual proteins of the cardiac system were reconstituted under controlled conditions. By this approach, myosin plus actin and troponin-tropomyosin from the normal and diabetic animals could be studied enzymatically. The proteins were isolated from the cardiac muscle of control and STZ-diabetic (4 weeks) rats. Sodium dodecyl sulfate gel electrophoretic patterns demonstrate differences in the cardiac TnT and TnI regions of diabetic animals suggesting the different amounts of TnT and/or TnI or possibly different cardiac isozymes in the regulatory protein complex. Myofibrils probed with a monoclonal antibody TnI-1 (specific for adult cardiac TnI) show a downregulation of cardiac TnI in diabetics when compared to its controls. Enzymatic data confirm a diminished calcium sensitivity in the regulation of the cardiac actomyosin system when regulatory protein(s) complex was recombined from diabetic hearts. Actomyosin ATPase activity in the hearts of diabetic animals was partially reversed when myosin from diabetic rats was regulated with the regulatory protein complex isolated from control hearts. To our knowledge, this is the first study which demonstrates that the regulatory proteins from normal hearts can upregulate cardiac myosin isolated from a pathologic rat model of diabetes. This diminished calcium sensitivity along with shifts in cardiac myosin heavy chain (V1-->V3) may be partially responsible for the impaired cardiac function in the hearts of chronic diabetic rats.

Expression of regulated cardiac troponin I in Escherichia coli.

The study of the functional effects of troponin isoform changes would be greatly aided by the development of a strategy permitting protein engineering and mutational analysis. To assess the role of troponin isoforms in regulating myofibrillar ATPase activity, we have expressed rat cardiac troponin I (cTnI) in E. coli and purified the protein to near homogeneity. We utilized the inducible expression vector pGEX-KG to create a glutathione-S-transferase fusion protein which can be cleaved with thrombin. Approximately 6 mg of cTnI can be purified from 1 l of culture. Ca2+Mg2+ ATPase activity was measured using the bacterially synthesized cTnI and the remaining components of the regulated actomyosin complex (troponin T, troponin C, tropomyosin, actin, and myosin) purified to homogeneity from mammalian hearts. In the presence of free Ca2+ ranging from 10(-2) to 10(-8) M, bacterially synthesized cTnI exhibits specific activity similar to that observed for control cTnI isolated from rat hearts. The bacterially synthesized protein is capable of stoichiometric phosphorylation and demonstrates appropriately regulated specific activity. These results establish the feasibility of using bacterial expression to study functional consequences of changes in expression of troponin isoforms.

Cardiac adaptations to chronic exercise in mice.

Transgenic mice can be created to serve as models of human cardiac disease. Despite the technology available to manipulate the cardiovascular system of the mouse, there is relatively little information available concerning the normal physiology of the mouse heart. Therefore, we have characterized the response of the adult mouse to chronic physical conditioning by swimming. Adult female C57/B16 mice were conditioned by swimming up to 90 min twice daily for 4 wk, resulting in a 10% increase in heart weight and a 16% increase in heart weight-to-body weight ratios compared with sedentary controls. The heart rate response to a submaximal work load decreased > 20% with this conditioning program. Succinate dehydrogenase activity increased markedly in the soleus muscles of the conditioned animals, from 28 +/- 3 to 44 +/- 3 nmol.mg-1.min-1. In contrast to these changes, which also characterize the exercise model in the rat, no increase in cardiac tissue norepinephrine content or in cardiac myosin or myofibrillar adenosinetriphosphatase (ATPase) activities was observed, and no change in the V1 predominant myosin isoform or alpha-myosin heavy chain mRNA profiles was seen in the hearts of the swimmers. This study establishes that mice are able to develop cardiac hypertrophy in response to chronic conditioning which is not associated with changes in the ATPase activities of cardiac muscle. These data should be of use to investigators using murine models to define the molecular basis of adaptive cardiac hypertrophy in vivo.

Prevention of age-related V1 myosin isozyme decrement in the adult rat heart.

The present investigation was undertaken to determine whether nifedipine could modulate the age-related relative decline in V1 myosin isozyme. Adult rats at age 24 weeks were treated with nifedipine. Myosin isozyme levels (V1, V2, and V3) at age 36 weeks, after 12 weeks of treatment with nifedipine, were compared with age-matched controls without nifedipine. As a natural effect of aging, the V1 percent was reduced from 63 +/- 6 at 16 wks to 29 +/- 3 at 36 wks (P < 0.001). In the 36 week treated Wistar-Kyoto (WKY) rats, the V1 level was 62 +/- 4, which was 47% higher than the age-matched untreated WKY. The V1 content of 36-week treated rats was the same as that of 16-week untreated rats. We conclude that the myosin isozyme changes occurring with aging can be prevented with nifedipine begun in the young adult rat.