Amino acid sequence of the human myeloma lambda chain Win.

The complete amino acid sequence of the variable region of a human myeloma immunoglobulin light chain (Win) has been determined. The sequence of the constant region has been verified by compositional analysis of its tryptic peptides. The amino acid sequence of the light chain Win corresponds to sub group II and shows no unusual amino acid replacements in its constant or variable regions when compared to other human gamma chains.

FDA perspective on peptide formulation and stability issues.

Traditionally, peptide drugs are prepared as sterile solutions and administered to patients by daily injection. However, this form of drug delivery causes pain and inconvenience to patients and thus has been poorly accepted. In addition to improving patient compliance, many novel delivery systems have been developed to address the need for prolonged, localized (targeted), or pulsatile drug action. Examples include, but are not limited to oral, nasal, or long-acting controlled release injectable dosage forms; a number of them have been approved by FDA recently. The unique characteristics and the relevant regulatory issues with respect to each type of delivery system are presented.

Rapid detection of herpes simplex virus by polymerase chain reaction.

This study reports the successful use of three sets of primers, each from different genes on the herpes simplex virus (HSV) genome, 1) the DNA polymerase gene, 2) the glycoprotein B gene, and 3) the glycoprotein D gene, for detection of HSV DNA by polymerase chain reaction (PCR). All three sets of primers detected the same HSV DNA in the throat and genital specimens. Using the conventional viral culture as a standard, PCR provided a sensitivity of 100% and a specificity of 100% in this study. In addition, a nested-PCR protocol using two sets of primers in the glycoprotein D gene, one set internal to the other, was evaluated for the amplification of HSV DNA in cerebrospinal fluid (CSF) from patients suspected of having herpes simplex encephalitis (HSE). Five of the 10 CSF specimens tested were found positive. In conclusion, PCR detection is a valuable tool for rapid diagnosis of HSV infection, especially for CSF specimens.

SC5b-9 is the most sensitive marker in assessing disease activity in Brazilian SLE patients.

This study investigated whether increased plasma levels of terminal complement complex (SC5b-9) or split products correlate with disease activity and clinical manifestations in Brazilian systemic lupus erythematosus (SLE) patients. Comparisons with conventional measurements of complement and other inflammatory markers were also performed. Plasma levels of SC5b-9, C3a desArg, C1rs-C1Inhibitor, C3b(Bb)P, C3, C4, erythrocyte sedimentation rate (ESR) and mucoproteins (MP) were measured in 41 patients with SLE of different disease activity: 10 patients with none, 15 patients with mild, and 16 patients with moderate or severe activity. All parameters, with the exception of C3 and C3b(Bb)P, showed a statistically significant correlation with disease activity. Plasma levels of SC5b-9, C3a desArg, C4, CH50, ESR and MP revealed significant differences between the groups of patients without activity and those with moderate or severe disease. Although none of the variables were able to discriminate between patients without and those with mild activity, SC5b-9, C3a desArg, C4, ESR and mucoproteins showed significant differences between the patients with mild and those with moderate or severe disease. Among all the variables, SC5b-9 levels showed the most significant results and correlated well with the severity of the disease (p < 0.0005). Our data suggest that elevated levels of complement activation products, particularly of SC5b-9 are more sensitive markers in assessing disease activity than conventional laboratory diagnosis. Modern complement diagnosis is therefore recommended for monitoring disease progress in SLE patients.

Clinical, autoimmune and demographic profile in systemic lupus erythematosus (SLE) patients from southern Brazil.

With the purpose of determining the association of clinical, autoimmune and demographic features, a group of 90 SLE patients from Southern Brazil were investigated. At diagnosis, 24% of them were under 20 years, 63% were between 20 and 40 years and 13% were older than 40 years. According to the ethnic background, there were 66% Brazilian-white patients, 21% Caucasians and 13% Mullatos/Blacks. Antinuclear antibodies (ANA) were present in 98%, anti-ds-DNA in 56% and anti-Sm in 31% of the patients. Anti-ds-DNA were more prevalent in the Caucasians (79%), while anti-Sm were increased in the Mullatos/Blacks (58%, p < 0.02) as compared to the white patients (Brazilian-whites = 22% and Caucasians = 42%). Neurologic involvement had lower prevalence in the group of Mullato/Black patients (8%) than in the Brazilian-whites (32%) and Caucasians (31%). Serositis was present in 51% of the Brazilian-whites, in 21% of the Caucasians and in 41% of the Mullatos/Blacks. On the other hand, the Mullato/ Black group had an increased prevalence of vasculitis (50%) and none of them presented with Raynaud's phenomenon. Younger patients at diagnosis presented higher frequency of renal involvement (p < 0.05), anti-ds-DNA positivity (p < 0.02) and more severe disease (p < 0.07), and in those patients diagnosed after age 40, 33% presented with Raynaud's phenomenon (p < 0.05). Regarding the anti-ds-DNA positivity, 78% of the patients had renal involvement (p < 0.01 RR 2.2) and 66% severe disease (p < 0.05). These results might be important in assessing clinical subsets and may aid individualized management of Brazilian SLE patients. Also, they may corroborate the need for special attention to racial composition in clinical and immunogenetic studies.

Genetic stability of rDNA production systems, a case report.

Producing rDNA proteins to be used as human therapeutic agents requires a biological production system during both storage and growth. The genetic make-up of a biological system is usually known through the laboratory history of the host strain and the process of vector construction. All master cell banks prepared for the production of medicinal products are fully characterized at the genetic and biochemical level. Whether these characteristics can be consistently maintained, particularly when the cells are propagated through a high number of generations and the culture grown into tens of thousands of litres, will have significant effects on the quality of the final products. Considerable experience and data regarding the stability of biological production systems involving plasmid and E. coli or yeast have been accumulated during the past 10 years. These data, including size and restriction analyses of the plasmid and sequence determination of relevant portions of the plasmid DNA isolated from cells collected before culture harvest, demonstrated that plasmid alteration at the structural level do occur, and some with high frequency or concentration. Assisted by such genetic information, criteria for accepting or rejecting a fermentation run can be established.

Regulatory concerns for the chemistry, manufacturing, and controls of oligonucleotide therapeutics for use in clinical studies.

It is important to remember that while a new class of therapeutic agents like oligonucleotides may introduce novel concerns, the basic regulatory issues regarding the chemistry, manufacturing, and controls of drug substances and drug products must be addressed. This article focuses on information that should be included in an Investigational New Drug Application (IND), a request to use an investigational drug in clinical studies. The regulatory challenge presented with oligonucleotide therapeutics is to prove the identity of the oligonucleotide, and demonstrate its quality, purity, and strength/potency using both those characteristics that are the same as all other drugs, as well as those that are unique. Most of the discussion will concern issues that are unique to oligonucleotides, or those topics that deserve more detailed attention than would be needed for more typical small molecule drugs. Regulatory issues will need to be evaluated so that safety concerns are addressed while not imposing undue burden on the sponsors of investigational drugs.

Amino acid sequence of the variable region of the light (lambda) chain from human myeloma cryoimmunoglobulin IgG Hil.

We have determined the complete amino acid sequence of the variable region of the light (lambda) chain from a human myeloma cryoimmunoglobulin (IgG Hil), the Fab fragment from which has been previously crystallized. The presence of unblocked alpha-amino terminal residue and the isolation of a CNBr fragment starting at position 46 and of a maleylated tryptic fragment spanning residues 61 to 189 provided three suitable starting points for automatic Edman degradation. In addition, tryptic peptides and chymotryptic subpeptides covering the whole extension of the light chain were obtained and characterized to further verify the sequence of the variable region and the established sequence of the constant region. The proposed sequence of the variable region indicates that it may be assigned to subgroup III. Positions 152 (serine) and 189 (arginine) correspond to the isotypic markers Kern- and Oz-, respectively. In addition, a novel substitution has been detected in the constant region where at position 155 isoleucine replaces the usually occurring valine.

Conformational transitions of synthetic DNA sequences with inserted bases, related to the dodecamer d(CGCGAATTCGCG).

Conformational transitions for a series of imperfect palindromes related to the dodecamer d(CGCGAATTCGCG) have been investigated. These sequences are: two isomeric 13-mers - d(CGCAGAATTCGCG) (13-merI) and d(CGCGAATTACGCG) (13-merII), 17-mer d(CGCGCGAATTACGCGCG) and 15-mer d(CGCGAAATTTACGCG). Insertion of a single adenine nucleotide prevents these sequences from being self-complementary. Analysis of thermodynamic parameters derived from the melting profiles together with other data at higher concentrations (NMR and calorimetry) indicates that the insertion of the additional nucleotide which lacks a complement in the opposite strand does not change the enthalpy of the duplex formation, but does alter the number of stable nucleation configurations. The relative position of the insertion within the self-complementary sequence determines the equilibrium between the duplex form and the single-stranded hairpin loop. C-G segments separated by the insertion from the rest of the molecule can undergo an independent conformational transition at high salt concentration, probably to the Z form.